Methods of monitoring immunosuppressive therapies in a transplant recipient

ABSTRACT

The present disclosure relates to methods of monitoring the status of an allograft in a transplant recipient, as well as to methods of monitoring and adjusting immunosuppressive therapies being administered to the transplant recipient.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is a Continuation of U.S. Non-Provisional applicationSer. No. 14/658,061, filed on Mar. 13, 2015, which claims the benefit ofU.S. Provisional Application No. 61/953,582 filed on Mar. 14, 2014,which are both incorporated herein by reference in their entirety.

FIELD

The present disclosure relates to methods of monitoring the status of anallograft in a transplant recipient, as well as to methods of monitoringand adjusting immunosuppressive therapies being administered to thetransplant recipient.

BACKGROUND

The immune system plays a defensive role in subjects, such as a humanindividual, but can also cause diseases, disorders, and otherundesirable conditions. In the case of medically intendedtransplantation of non-self (allograft) cells, tissues, or organs intoan individual, the recipient's immune system recognizes the allograft tobe foreign to the body and activates various mechanisms to reject theallograft. Thus, it is necessary to medically suppress the normal immunesystem responses to reject the transplant. The medical practice ofimmunosuppression in transplant recipients has evolved to include aregimen of prophylactic pharmacologic agents, typically beginning withinduction therapies to deplete lymphocytes, followed by maintenancedrugs intended to inhibit activation or replication of lymphocytes suchas corticosteroids, calcineurin inhibitors (such as tacrolimus), andadditional inhibitors of lymphocyte replication (such as mycophenolatemofetil). Changing or varying the amount of immunosuppressive drugsadministered to a transplant recipient has largely been guided byempirical experience. After transplant, the dosage ofimmunosuppressant(s) are reduced over time to reduce the incidence andseverity of side effects, such as increased risk of infectious diseases,while still avoiding immune rejection of the allograft.

After transplantation, the status of the allograft in the transplantrecipient may be monitored for the remainder of his/her lifetime,including assessment function of the allograft and immune-mediatedrejection of the allograft. In heart transplantation, for example,surveillance for rejection may include up to 15 scheduled biopsieswithin the first year of the transplant to provide specimens of theheart muscle for histologic evaluation by a pathologist. Each biopsyprocedure is invasive (percutaneous passage of a transvenous catheterinto the right ventricle of the heart), stressful, inconvenient, andincumbent of procedural risks for the patient, as well as beingexpensive. Moreover, the biopsy sampling is extremely localized, sohistological abnormalities in any non-biopsied areas of the heart aremissed. The grading of biopsies is subjective, and discordance of biopsyfindings is common between independent pathologists. In the standardclinical care of transplant recipients, there are a variety of clinicallaboratory diagnostics tests, in addition to periodic biopsies, thatprovide some information relating to the status of the allograft. Forexample, the serum trough levels of the calcineurin inhibitor drug aremeasured to estimate adequacy of intended coverage. Other assays detectthe presence of antibodies directed against the allograft. Biopsy isprimarily used for surveillance of transplant rejection within the firstyear, but this invasive method is not well suited or established forguiding individualized immunosuppressive therapy in the longer term (e.gbeyond one year after transplant) maintenance care of patients.Non-invasive gene expression methods inform on the status of the immunesystem by examining the status of genes expressed in immune cells.AlloMap Molecular Expression Testing is an FDA-cleared test availablefor heart transplant recipients. Tests are in development for monitoringother solid organ transplants.

In addition to existing invasive biopsy methods of monitoring transplantstatus, there are currently no specific tests with a demonstratedability to guide individualization (and further minimization) ofimmunosuppressive drugs for long-term maintenance of a transplantrecipient. Data, mostly derived from registry studies, have identifiedcertain clinical risk factors for transplant loss or death such asrecipient age, gender, and race, as well as donor features such as coldischemia, time, and age. However, determining these clinical riskfactors does not supplant the need for individualized treatment androutine surveillance of transplant recipients.

There exists a need for improved noninvasive methods of diagnosing andmonitoring that status of an allograft in a transplant recipient, aswell as for methods of determining the need to adjust immunosuppressivetherapy being administered to a transplant recipient.

BRIEF SUMMARY

In one aspect, the present disclosure relates to methods of monitoringimmunosuppressive therapy in a subject, the method including: a)providing cell-free DNA from a sample obtained from a subject who is therecipient of an organ transplant from a donor, b) sequencing a panel ofsingle nucleotide polymorphisms (SNPs) from the cell-free DNA, where thepanel of SNPs is suitable for differentiating between donor-derivedcell-free DNA and recipient-derived cell-free DNA, c) assaying variancein SNP allele distribution patterns in the panel as compared to expectedhomozygous or heterozygous distribution patterns to determine the levelof donor-derived cell-free DNA, and d) diagnosing the status of thetransplanted organ in the subject, where a change in levels or varianceof the donor-derived cell-free DNA over a time interval is indicative oftransplanted organ status and a basis for adjusting immunosuppressivetherapy.

In another aspect, the present disclosure relates to methods ofadjusting an immunosuppressive therapy in a subject, the methodincluding: a) providing cell-free DNA from a sample obtained from asubject who is the recipient of an organ transplant from a donor, b)sequencing a panel of single nucleotide polymorphisms (SNPs) from thecell-free DNA, where the panel of SNPs is suitable for differentiatingbetween donor-derived cell-free DNA and recipient-derived cell-free DNA,c) assaying variance in SNP allele distribution patterns in the panel ascompared to expected homozygous or heterozygous distribution patterns todetermine the level of donor-derived cell-free DNA, d) diagnosing thestatus of the transplanted organ in the subject, where a change inlevels or variance of the donor-derived cell-free DNA over a timeinterval is indicative of transplanted organ status, e) adjustingimmunosuppressive therapy being administered to the subject.

In some embodiments that may be combined with any of the precedingembodiments, an increase in the levels or variance of the donor-derivedcell-free DNA over the time interval is indicative of transplantrejection, a need for adjusting immunosuppressive therapy, and/or a needfor further investigation of the transplanted organ status. In someembodiments that may be combined with any of the preceding embodiments,a decrease in the levels or variance of the donor-derived cell-free DNAover the time interval is indicative of transplant tolerance, a need foradjusting immunosuppressive therapy, and/or a need for furtherinvestigation of the transplanted organ status. In some embodiments thatmay be combined with any of the preceding embodiments, no change in thelevels or variance of the donor-derived cell-free DNA over the timeinterval is indicative of stable transplant rejection status and/oropportunity for adjusting immunosuppressive therapy. In some embodimentsthat may be combined with any of the preceding embodiments,immunosuppressive therapy being administered to the subject isincreased. In some embodiments that may be combined with any of thepreceding embodiments, immunosuppressive therapy being administered tothe subject is decreased. In some embodiments that may be combined withany of the preceding embodiments, immunosuppressive therapy beingadministered to the subject is maintained. In some embodiments that maybe combined with any of the preceding embodiments, the organ transplantis a kidney transplant. In some embodiments that may be combined withany of the preceding embodiments, the organ transplant is a hearttransplant. In some embodiments that may be combined with any of thepreceding embodiments, the sample is a plasma sample. In someembodiments that may be combined with any of the preceding embodiments,the panel of SNPs includes at least 20 independent SNPs. In someembodiments that may be combined with any of the preceding embodiments,the panel of SNPs includes independent SNPs selected from rs1004357,rs10092491, rs1019029, rs1027895, rs10488710, rs10500617, rs1058083,rs10768550, rs10773760, rs10776839, rs1109037, rs12480506, rs1294331,rs12997453, rs13134862, rs13182883, rs13218440, rs1336071, rs1358856,rs1410059, rs1478829, rs1490413, rs1498553, rs1523537, rs1554472,rs159606, rs1736442, rs1821380, rs1872575, rs2046361, rs2073383,rs214955, rs2175957, rs221956, rs2255301, rs2269355, rs2270529,rs2272998, rs2291395, rs2292972, rs2342747, rs2399332, rs2503107,rs2567608, rs279844, rs2811231, rs2833736, rs2920816, rs315791,rs321198, rs338882, rs3744163, rs3780962, rs4288409, rs430046,rs4364205, rs445251, rs4530059, rs4606077, rs464663, rs4789798,rs4796362, rs4847034, rs521861, rs560681, rs5746846, rs576261, rs590162,rs6444724, rs6591147, rs6811238, rs689512, rs6955448, rs7041158,rs7205345, rs722290, rs7229946, rs740598, rs7520386, rs7704770,rs8070085, rs8078417, rs891700, rs901398, rs9546538, rs9606186,rs985492, rs9866013, rs987640, rs9905977, rs993934, and rs9951171. Insome embodiments that may be combined with any of the precedingembodiments, sequencing the panel of SNPs is performed using a multiplexsequencing platform. In some embodiments that may be combined with anyof the preceding embodiments, the time interval is about 12-14 monthsafter the transplant from the donor to the recipient subject occurred.In some embodiments that may be combined with any of the precedingembodiments, the method further includes testing for viral load. In someembodiments, the testing includes determining the presence of a virusselected from CMV, EBV, anellovirus, and BKV. In some embodiments thatmay be combined with any of the preceding embodiments, the methodfurther includes conducting one or more gene expression profilingassays. In some embodiments, the gene expression profiling assay is anAlloMap test.

In one aspect, the present disclosure relates to methods of monitoringthe status of a transplanted organ in a subject, the method including:a) providing cell-free DNA from a sample obtained from a subject who isthe recipient of an organ transplant from a donor, b) sequencing a panelof single nucleotide polymorphisms (SNPs) from the cell-free DNA, wherethe panel of SNPs is suitable for differentiating between donor-derivedcell-free DNA and recipient-derived cell-free DNA, c) assaying variancein SNP allele distribution patterns in the panel as compared to expectedhomozygous or heterozygous distribution patterns to determine the levelof donor-derived cell-free DNA, and d) diagnosing the status of thetransplanted organ in the subject, where a change in levels or varianceof the donor-derived cell-free DNA over a time interval is indicative ofthe status of the transplanted organ.

In another aspect, the present disclosure relates to methods ofmonitoring immunosuppressive therapy in a subject, the method including:a) providing cell-free DNA from a sample obtained from a subject who isthe recipient of an organ transplant from a donor, b) sequencing a panelof single nucleotide polymorphisms (SNPs) from the cell-free DNA, wherethe panel of SNPs is suitable for differentiating between donor-derivedcell-free DNA and recipient-derived cell-free DNA, c) assaying variancein SNP allele distribution patterns in the panel as compared to expectedhomozygous or heterozygous distribution patterns to determine the levelof donor-derived cell-free DNA, and d) diagnosing the status of thetransplanted organ in the subject, where a change in levels or varianceof the donor-derived cell-free DNA over a time interval is indicative oftransplanted organ status and a basis for adjusting immunosuppressivetherapy.

In another aspect, the present disclosure relates to methods ofadjusting an immunosuppressive therapy in a subject, the methodincluding: a) providing cell-free DNA from a sample obtained from asubject who is the recipient of an organ transplant from a donor, b)sequencing a panel of single nucleotide polymorphisms (SNPs) from thecell-free DNA, where the panel of SNPs is suitable for differentiatingbetween donor-derived cell-free DNA and recipient-derived cell-free DNA,c) assaying variance in SNP allele distribution patterns in the panel ascompared to expected homozygous or heterozygous distribution patterns todetermine the level of donor-derived cell-free DNA, d) diagnosing thestatus of the transplanted organ in the subject, where a change inlevels or variance of the donor-derived cell-free DNA over a timeinterval is indicative of transplanted organ status, and e) adjustingimmunosuppressive therapy being administered to the subject.

In some embodiments that may be combined with any of the precedingembodiments, an increase in the levels or variance of the donor-derivedcell-free DNA over the time interval is indicative of transplantrejection, a need for adjusting immunosuppressive therapy, and/or a needfor further investigation of the transplanted organ status. In someembodiments that may be combined with any of the preceding embodiments,a decrease in the levels or variance of the donor-derived cell-free DNAover the time interval is indicative of transplant tolerance, a need foradjusting immunosuppressive therapy, and/or a need for furtherinvestigation of the transplanted organ status. In some embodiments thatmay be combined with any of the preceding embodiments, no change in thelevels or variance of the donor-derived cell-free DNA over the timeinterval is indicative of stable transplant rejection status and/oropportunity for adjusting immunosuppressive therapy. In some embodimentsthat may be combined with any of the preceding embodiments,immunosuppressive therapy being administered to the subject isincreased. In some embodiments that may be combined with any of thepreceding embodiments, immunosuppressive therapy being administered tothe subject is decreased. In some embodiments that may be combined withany of the preceding embodiments, immunosuppressive therapy beingadministered to the subject is maintained. In some embodiments that maybe combined with any of the preceding embodiments, the organ transplantis a kidney transplant. In some embodiments that may be combined withany of the preceding embodiments, the organ transplant is a hearttransplant. In some embodiments that may be combined with any of thepreceding embodiments, the organ transplant is selected from a livertransplant, a lung transplant, and a pancreas transplant. In someembodiments that may be combined with any of the preceding embodiments,the sample is a plasma sample. In some embodiments that may be combinedwith any of the preceding embodiments, the panel of SNPs includesindependent SNPs selected from rs1004357, rs10092491, rs1019029,rs1027895, rs10488710, rs10500617, rs1058083, rs10768550, rs10773760,rs10776839, rs1109037, rs12480506, rs1294331, rs12997453, rs13134862,rs13182883, rs13218440, rs1336071, rs1358856, rs1410059, rs1478829,rs1490413, rs1498553, rs1523537, rs1554472, rs159606, rs1736442,rs1821380, rs1872575, rs2046361, rs2073383, rs214955, rs2175957,rs221956, rs2255301, rs2269355, rs2270529, rs2272998, rs2291395,rs2292972, rs2342747, rs2399332, rs2503107, rs2567608, rs279844,rs2811231, rs2833736, rs2920816, rs315791, rs321198, rs338882,rs3744163, rs3780962, rs4288409, rs430046, rs4364205, rs445251,rs4530059, rs4606077, rs464663, rs4789798, rs4796362, rs4847034,rs521861, rs560681, rs5746846, rs576261, rs590162, rs6444724, rs6591147,rs6811238, rs689512, rs6955448, rs7041158, rs7205345, rs722290,rs7229946, rs740598, rs7520386, rs7704770, rs8070085, rs8078417,rs891700, rs901398, rs9546538, rs9606186, rs985492, rs9866013, rs987640,rs9905977, rs993934, and rs9951171. In some embodiments that may becombined with any of the preceding embodiments, the panel of SNPsincludes SNPs that have an overall population minor allele frequencyof >0.4, a target population minor allele frequency of >0.4, the lowestpolymerase error rate of the 6 potential allele transitions ortransversions, and the genomic distance between each independent SNPis >500 kb. In some embodiments that may be combined with any of thepreceding embodiments, the panel of SNPs includes independent SNPsselected from rs10488710, rs279844, rs1048290, rs1049379, rs1051614,rs1052637, rs1055851, rs1056033, rs1056149, rs1064074, rs1078004,rs10831567, rs6811238, rs11106, rs11210490, rs1126899, rs1127472,rs1127893, rs1130857, rs1049544, rs11547806, rs12237048, rs430046,rs12508837, rs12529, rs12717, rs13184586, rs13295990, rs13428, rs13436,rs1374570, rs14080, rs1411271, rs576261, rs14155, rs1151687, rs1565933,rs1600, rs1678690, rs1881421, rs1897820, rs1898882, rs2056844, rs20575,rs10092491, rs2070426, rs2071888, rs2075322, rs2180314, rs2185798,rs2227910, rs2228560, rs2229571, rs2229627, rs2245285, rs2342747,rs2248490, rs2253592, rs2254357, rs2275047, rs2279665, rs2279776,rs2281098, rs2287813, rs4364205, rs2289751, rs2289818, rs2292830,rs2294092, rs2295005, rs2296545, rs2297236, rs2302443, rs2306049,rs1022478, rs445251, rs230898, rs231235, rs2342767, rs236152, rs2362450,rs2384571, rs2455230, rs246703, rs2480345, rs248385, rs2498982,rs2505232, rs2509943, rs2519123, rs2523072, rs2571028, rs2657167,rs28686812, rs2946994, rs1294331, rs10419826, rs3088241, rs3110623,rs3173615, rs3190321, rs3205187, rs344141, rs35596415, rs362124,rs36657, rs1872575, rs159606, rs3731877, rs3734311, rs3735615,rs3740199, rs3748930, rs3751066, rs3790993, rs3802265, rs3803763,rs1004357, rs3803798, rs3809972, rs3810483, rs3812571, rs3813609,rs3814182, rs3816800, rs3826709, rs3829655, rs3951216, rs1019029,rs408600, rs41317515, rs436278, rs448012, rs475002, rs4845480,rs4849167, rs4865615, rs1027895, rs4890012, rs492594, rs4940019,rs4971514, rs523104, rs528557, rs545500, rs561930, rs57010808,rs57285449, rs10500617, rs6061243, rs609521, rs62490396, rs625223,rs638405, rs6459166, rs648802, rs6510057, rs6764714, rs10768550,rs6790129, rs6794, rs6807362, rs6838248, rs713598, rs7161563, rs726009,rs7289, rs7301328, rs7332388, rs10773760, rs743616, rs743852, rs745142,rs7451713, rs7526132, rs7543016, rs7601771, rs7785899, rs7825,rs8009219, rs10776839, rs8025851, rs8058696, rs8076632, rs8097,rs8103906, rs874881, rs9262, rs9289122, rs936019, rs9393728, rs1109037,rs977070, rs9865242, rs12480506, rs560681, rs12997453, rs13134862,rs13218440, rs1358856, rs1410059, rs1478829, rs1498553, rs1523537,rs4606077, rs1554472, rs1736442, rs1821380, rs2046361, rs214955,rs2175957, rs2255301, rs2269355, rs2270529, rs2272998, rs2291395,rs2292972, rs2399332, rs2503107, rs2567608, rs2811231, rs2833736,rs315791, rs321198, rs6955448, rs338882, rs3780962, rs4288409,rs4530059, rs464663, rs4789798, rs4796362, rs4847034, rs521861,rs1058083, rs5746846, rs590162, rs6444724, rs6591147, rs689512,rs7205345, rs722290, rs740598, rs7520386, rs221956, rs7704770,rs8070085, rs8078417, rs891700, rs901398, rs9546538, rs9606186,rs985492, rs9866013, rs987640, rs13182883, rs9905977, rs993934,rs9951171, rs10274334, rs10421285, rs1043413, rs1044010, rs1045248,rs1045644, and rs1047979. In some embodiments, the SNP panel includesabout 195 to about 200, about 200 to about 205, about 210 to about 215,about 215 to about 220, about 220 to about 225, about 225 to about 230,about 230 to about 235, about 235 to about 240, about 240 to about 245,about 245 to about 250, about 250 to about 255, about 255 to about 260,about 260 to about 265, or about 260 to about 266 of the independentSNPs. In some embodiments that may be combined with any of the precedingembodiments, the SNP panel includes rs10488710, rs279844, rs1048290,rs1049379, rs1051614, rs1052637, rs1055851, rs1056033, rs1056149,rs1064074, rs1078004, rs10831567, rs6811238, rs11106, rs11210490,rs1126899, rs1127472, rs1127893, rs1130857, rs1049544, rs11547806,rs12237048, rs430046, rs12508837, rs12529, rs12717, rs13184586,rs13295990, rs13428, rs13436, rs1374570, rs14080, rs1411271, rs576261,rs14155, rs1151687, rs1565933, rs1600, rs1678690, rs1881421, rs1897820,rs1898882, rs2056844, rs20575, rs10092491, rs2070426, rs2071888,rs2075322, rs2180314, rs2185798, rs2227910, rs2228560, rs2229571,rs2229627, rs2245285, rs2342747, rs2248490, rs2253592, rs2254357,rs2275047, rs2279665, rs2279776, rs2281098, rs2287813, rs4364205,rs2289751, rs2289818, rs2292830, rs2294092, rs2295005, rs2296545,rs2297236, rs2302443, rs2306049, rs1022478, rs445251, rs230898,rs231235, rs2342767, rs236152, rs2362450, rs2384571, rs2455230,rs246703, rs2480345, rs248385, rs2498982, rs2505232, rs2509943,rs2519123, rs2523072, rs2571028, rs2657167, rs28686812, rs2946994,rs1294331, rs10419826, rs3088241, rs3110623, rs3173615, rs3190321,rs3205187, rs344141, rs35596415, rs362124, rs36657, rs1872575, rs159606,rs3731877, rs3734311, rs3735615, rs3740199, rs3748930, rs3751066,rs3790993, rs3802265, rs3803763, rs1004357, rs3803798, rs3809972,rs3810483, rs3812571, rs3813609, rs3814182, rs3816800, rs3826709,rs3829655, rs3951216, rs1019029, rs408600, rs41317515, rs436278,rs448012, rs475002, rs4845480, rs4849167, rs4865615, rs1027895,rs4890012, rs492594, rs4940019, rs4971514, rs523104, rs528557, rs545500,rs561930, rs57010808, rs57285449, rs10500617, rs6061243, rs609521,rs62490396, rs625223, rs638405, rs6459166, rs648802, rs6510057,rs6764714, rs10768550, rs6790129, rs6794, rs6807362, rs6838248,rs713598, rs7161563, rs726009, rs7289, rs7301328, rs7332388, rs10773760,rs743616, rs743852, rs745142, rs7451713, rs7526132, rs7543016,rs7601771, rs7785899, rs7825, rs8009219, rs10776839, rs8025851,rs8058696, rs8076632, rs8097, rs8103906, rs874881, rs9262, rs9289122,rs936019, rs9393728, rs1109037, rs977070, rs9865242, rs12480506,rs560681, rs12997453, rs13134862, rs13218440, rs1358856, rs1410059,rs1478829, rs1498553, rs1523537, rs4606077, rs1554472, rs1736442,rs1821380, rs2046361, rs214955, rs2175957, rs2255301, rs2269355,rs2270529, rs2272998, rs2291395, rs2292972, rs2399332, rs2503107,rs2567608, rs2811231, rs2833736, rs315791, rs321198, rs6955448,rs338882, rs3780962, rs4288409, rs4530059, rs464663, rs4789798,rs4796362, rs4847034, rs521861, rs1058083, rs5746846, rs590162,rs6444724, rs6591147, rs689512, rs7205345, rs722290, rs740598,rs7520386, rs221956, rs7704770, rs8070085, rs8078417, rs891700,rs901398, rs9546538, rs9606186, rs985492, rs9866013, rs987640,rs13182883, rs9905977, rs993934, rs9951171, rs10274334, rs10421285,rs1043413, rs1044010, rs1045248, rs1045644, and rs1047979. In someembodiments that may be combined with any of the preceding embodiments,sequencing the panel of SNPs is performed using a multiplex sequencingplatform. In some embodiments that may be combined with any of thepreceding embodiments, the level of donor-derived cell-free DNA in thesample is determined without using genotype information. In someembodiments that may be combined with any of the preceding embodiments,the method further includes testing for the presence of an infectiousagent. In some embodiments that may be combined with any of thepreceding embodiments, the infectious agent is selected from viruses,bacteria, fungi, and parasites. In some embodiments, the viruses areselected from Cytomegalovirus, Epstein-Barr virus, Anelloviridae, and BKvirus. In some embodiments that may be combined with any of thepreceding embodiments, the method further includes conducting one ormore gene expression profiling assays. In some embodiments, acombination score is calculated based on the results of the level ofdonor-derived cell-free DNA and the results of the gene expressionprofiling assay. In some embodiments that may be combined with any ofthe preceding embodiments, the gene expression profiling assay is anAlloMap test.

DESCRIPTION OF THE FIGURES

FIG. 1 illustrates that donor-derived cell-free DNA (dd-cfDNA) isgreater in plasma taken from heart transplant recipients experiencingacute cellular rejection than from those not experiencing rejection.Data was generated using plasma from heart transplant recipientscollected in PPT tubes. Patient samples were assigned to NR(non-rejection) and R (rejection) categories based on the status ofendomyocardial biopsy performed at the same patient visit. Donor-derivedcell-free DNA is expressed as a percent of the total cell-free DNA,measured as described herein. Groups differ by t-test (P=0.017).

FIG. 2 illustrates that dd-cfDNA is elevated prior to heart transplantrejection. Data was generated using plasma from heart transplantrecipients collected in PPT tubes. Patient samples were assigned R(rejection) status based on the status of endomyocardial biopsyperformed at the same patient visit. Visits prior to the rejection eventwere grouped according to the time prior to the rejection visit (25 forfewer days or longer than 25 days). Donor-derived cell-free DNA isexpressed as a percent of the total cell-free DNA, measured as describedherein.

FIG. 3 illustrates that dd-cfDNA is reduced following treatment of hearttransplant recipients for acute cellular rejection with increasedimmunosuppressive therapy. Data was generated using plasma from hearttransplant recipients collected in PPT tubes. Patient sample wasassigned R (rejection, triangle), or NR (non-rejection, circle) statusbased on the endomyocardial biopsy performed at the same patient visit.Donor-derived cell-free DNA is expressed as a percent of the totalcell-free DNA, measured as described herein. This patient receivedlarge-dose immunosuppression with prednisone based on the endomyocardialbiopsy status of 2R rejection (index event at 225 days post-transplant).

FIG. 4 illustrates that dd-cfDNA is reduced following treatment ofkidney transplant recipients for acute cellular rejection with increasedimmunosuppressive therapy. Data was generated using plasma from kidneytransplant recipients collected as supernatant from CPT processing.Patient sample was assigned R (rejection, triangle) based on the biopsyresults, NR (non-rejection, circles) based on low serum creatinine and.Donor-derived cell-free DNA is expressed as a percent of the totalcell-free DNA, measured as described herein.

FIG. 5 illustrates dd-cfDNA values from Streck BCT plasma collectiontubes following heart transplantation. Patient samples were blinded torejection status or outcome. Three subjects, three visits each, each adifferent shape symbol. Donor-derived cell-free DNA is expressed as apercent of the total cell-free DNA, measured as described herein.

FIG. 6A-FIG. 6C illustrates that dd-cfDNA is a signal unique from geneexpression profiling of blood markers, and the combination of both canbetter identify rejection. FIG. 6A illustrates dd-cfDNA levels fromheart transplant recipients. cfDNA data was generated using plasma fromheart transplant recipients collected in PPT tubes. Patient samples wereassigned R (rejection) status based on the status of endomyocardialbiopsy performed at the same patient visit. Donor-derived cell-free DNAis expressed as a percent of the total cell-free DNA, measured asdescribed herein. FIG. 6B illustrates AlloMap data from the transplantrecipients in FIG. 6A. Gene expression data was generated usingmononuclear cells collected using CPT tubes. Gene expression wasmeasured by AlloMap Molecular Expression Testing. FIG. 6C illustratescombined dd-cfDNA data and AlloMap data. The values for dd-cfDNA (FIG.6A) and AlloMap (FIG. 6B) were scaled to the same range and additivelycombined. The combined score is better at discriminating rejection fromnon-rejection than either cfDNA or gene expression alone, as measured bythe area under the curve of a receiver-operator characteristics plot.

FIG. 7 illustrates an exemplary workflow of the polymorphic markeranalysis methods described herein.

DETAILED DESCRIPTION

The following description is presented to enable a person of ordinaryskill in the art to make and use the various embodiments. Descriptionsof specific devices, techniques, and applications are provided only asexamples. Various modifications to the examples described herein will bereadily apparent to those of ordinary skill in the art, and the generalprinciples defined herein may be applied to other examples andapplications without departing from the spirit and scope of the variousembodiments. Thus, the various embodiments are not intended to belimited to the examples described herein and shown, but are to beaccorded the scope consistent with the claims.

The present disclosure relates to methods of monitoring the status of anallograft in a transplant recipient, as well as to methods of monitoringand adjusting immunosuppressive therapies being administered to thetransplant recipient.

Overview

The present disclosure is based, at least in part, on Applicant'sdevelopment of techniques for probing the status of an allograft in atransplant recipient. Transplant recipients contain an allograft that isforeign to the recipient's body. This triggers an immune response fromthe recipient's immune system, which may lead to acute and/or chronictransplant rejection. Applicant's methods involve the analysis ofcell-free DNA (cfDNA) from the transplant recipient to diagnose thestatus of the transplant and inform the need to adjust immunosuppressivetherapy being administered to the transplant recipient. Without wishingto be bound by theory, it is thought that transplant rejection isassociated with the death of cells in the transplanted (donor) organ ortissue, which will release donor-derived DNA from the dying donor cells,thus releasing donor-derived cell-free DNA (dd-cfDNA) into thebloodstream of the recipient. To assay the status of the allograft inthe recipient, cell-free DNA can be extracted from a sample from therecipient, such as a bodily fluid, and various polymorphic markers, suchas single nucleotide polymorphism (SNP) loci, can be sequenced, wherethe panel of polymorphic markers, such as a panel of SNPs, is suitablefor differentiating between donor-derived cell-free DNA andrecipient-derived cell-free DNA (rd-cfDNA). The specific polymorphicmarkers selected to be on the panel include those that are identified ashaving low probabilities of being identical in any two individuals, thusmaking them appropriate for differentiating between recipient-derivedcell free DNA and donor-derived cell-free DNA. The number of polymorphicmarkers on the panel such as, for example, the number of SNPs on thepanel, will be sufficient to discriminate between recipient and donoralleles even in related individuals (excepting identical twins). Theallele distribution patterns of polymorphic markers in the panel can beassayed to determine variance in the patterns as compared to expectedhomozygous (i.e. 0% or 100% of each allele) or heterozygous (i.e. 50% ofeach allele) distribution patterns, which can be used to determine thelevel of donor-derived cell-free DNA. Individual genotyping of the donorand the recipient to determine which allele of the polymorphic locusbelongs to the donor and/or the recipient is not necessary, as variancein the polymorphic marker allele distribution pattern from expectedhomozygous or heterozygous distribution patterns informs the presence ofdonor-derived cell-free DNA in the population of cell-free DNA moleculesisolated from the transplant recipient. In other words, it is assumedthat the majority signal from the cell-free DNA sample isrecipient-derived DNA and that the minority signal is donor-derived DNA,and this information can be used to calculate the levels ofdonor-derived DNA in the cell-free DNA sample. Changes in the levels orvariance of the donor-derived cell-free DNA over time can be used toinform the status of the allograft in the transplant recipient, as wellas inform a need to adjust or maintain an immunosuppressive therapybeing administered to the subject.

Accordingly, the present disclosure provides methods of monitoring thestatus of an allograft in a transplant recipient, as well as methods ofmonitoring and/or adjusting an immunosuppressive therapy being or to beadministered to a transplant recipient. Monitoring the status of anallograft involves analyzing various aspects which provide usefulinformation about the physiological state of the allograft such as, forexample, the level of donor-derived cell-free DNA in a sample from thetransplant recipient. The methods of the present disclosure may be usedto predict the risk of future transplant rejection such as, for example,the risk of rejection within the following 3-6 months after analysis ofsamples from the transplant recipient. The methods of the presentdisclosure may also be used to diagnose or predict the risk of allograftdysfunction, such as chronic renal insufficiency or cardiac allograftvasculopathy (CAV) (e.g. within the next 1-2 years after analysis ofsamples from the transplant recipient). The methods of the presentdisclosure may also be used provide an assessment of the immune statusof the transplant recipient, which may be used to guide decisionsregarding immunosuppressive therapy in the transplant recipient. Themethods of the present disclosure may also be used to guide decisionsrelated to adjustment of immunosuppressive therapies being administeredto the transplant recipient. Additional benefits and/or uses of themethods of the present disclosure will be readily apparent to one ofskill in the art.

Cell-Free DNA

The methods of the present disclosure involve the analysis of cell-freeDNA from a transplant recipient to diagnose the status of the transplantand/or to inform a need to adjust immunosuppressive therapy beingadministered to the transplant recipient. Cell-free DNA generally refersto DNA that is present outside of a cell such as, for example, DNA thatis present in a bodily fluid (e.g. blood, plasma, serum, etc.) of asubject. Cell-free DNA may have originated from various locations withina cell. Cell-free DNA may have originated from, for example, nuclear DNAand mitochondrial DNA. Without wishing to be bound by theory, it isbelieved that cell-free DNA is released from cells via apoptosis ornecrosis of the cells (i.e. cell death). Accordingly, and withoutwishing to be bound by theory, it is believed that during transplantrejection, apoptosis or necrosis of transplanted (donor) cells willresult in donor-derived cell-free DNA being released into the bodilyfluid of a transplant recipient. Transplant recipients undergoingtransplant rejection may then have a cell-free DNA population in theirbodily fluids which includes both their own endogenous cell-free DNA(recipient-derived cell-free DNA) as well as cell-free DNA thatoriginated from the donor (donor-derived cell-free DNA). Determining achange in the levels and/or variance in donor-derived cell-free DNA in atransplant recipient over time according to the methods of the presentdisclosure may be used to diagnose the status of the allograft andinform a need to adjust immunosuppressive therapy.

Cell-free RNA may also be collected from a transplant recipient andanalyzed by analogous methods as described above and also analysis ofrecipient RNA levels from specific marker genes to diagnose the statusof the transplant and/or to inform a need to adjust immunosuppressivetherapy being administered to the transplant recipient. Thus, themethods of the present disclosure generally relate to analysis ofcell-free nucleic acids from a transplant recipient to diagnose thestatus of the transplant and/or to inform a need to adjustimmunosuppressive therapy being administered to the transplantrecipient.

Subjects and Samples

The methods of the present disclosure involve providing cell-free DNAfrom a sample obtained from a subject who is the recipient of anallograft from a donor. In this sense, the subject is a transplantrecipient who contains an allograft from a donor, and is typically ahuman transplant recipient. The transplant recipient may have receivedone or more of a variety of allografts from a donor. Allografts mayinclude transplanted organs. Transplanted organs may include, forexample, a heart, a kidney, a lung, a liver, a pancreas, a cornea, anorgan system, or other solid organs. The transplant received by thetransplant recipient from the donor may also include other allograftssuch as, for example, a bone marrow transplant, pancreatic islet cells,stem cells, skin tissue, skin cells, or a xenotransplant.

The provided sample may include a bodily fluid isolated from thetransplant recipient. Samples obtained from the transplant recipientcontain cell-free DNA, and the total cell-free DNA present in the samplemay be entirely recipient-derived cell-free DNA, or the total cell-freeDNA present in the sample may include a mixture of recipient-derivedcell-free DNA and donor-derived cell-free DNA. Samples may include abodily fluid from the transplant recipient such as, for example, plasma,serum, whole blood, sweat, tears, saliva, ear flow fluid, sputum, fluidfrom bone marrow suspension, lymph fluid, urine, saliva, semen, vaginalflow, cerebrospinal fluid, brain fluid, ascites, milk, secretions of therespiratory, and intestinal or genitourinary tract fluids. In someembodiments where the sample is plasma, plasma derived from the venousblood of the transplant recipient can be obtained.

Once a sample is obtained, it can be used directly, frozen, or otherwisestored in a condition that maintains the integrity of the cell-free DNAfor short periods of time by preventing degradation and/or contaminationwith genomic DNA or other nucleic acids. The amount of a sample that istaken at a particular time may vary, and may depend on additionalfactors, such as any need to repeat analysis of the sample. In someembodiments, up to 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mLof a sample is obtained. In some embodiments, 1-50, 2-40, 3-30, or 4-20mL of sample is obtained. In some embodiments, more than 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 mL ofa sample is obtained.

Samples may be taken from a transplant recipient over a period of time(i.e. over a time interval). The time at which samples are taken fromthe transplant recipient following the transplant event may vary.Samples may be taken from a transplant recipient at various times andover various periods of time for use in determining the status of theallograft according to the methods of the present disclosure. Forexample, samples may be taken from the transplant recipient within daysand weeks after, about three months after, about six months after, aboutnine months after, or less than one year after the transplant event.Samples may be taken from the transplant recipient at various timesbefore the one year anniversary of the transplant event, at the one yearanniversary of the transplant event, or at various times after the oneyear anniversary of the transplant event. For example, at the one yearanniversary after a transplant, samples may begin to be taken from thetransplant recipient at month 12 (i.e. the one year anniversary of thetransplant event) and continue to be taken for periods of time afterthis. In some embodiments, the time period for obtaining samples from atransplant recipient is within the first few days after the transplantfrom the donor to the recipient occurred. This may be done to monitorinduction therapy. In some embodiments, the time period for obtainingsamples from a transplant recipient is during tapering of theimmunosuppressive regimen, a period that occurs during the first 12months after the transplant from the donor to the recipient occurred. Insome embodiments, the time period for obtaining samples from atransplant recipient is during the initial long term immunosuppressivemaintenance phase, beginning about 12-14 months after the transplantfrom the donor to the recipient occurred. In some embodiments, the timeperiod for obtaining samples from a transplant recipient is during theentire long term maintenance of the immunosuppressive regimen, any timebeyond 12 months after the transplant from the donor to the recipientoccurred.

Where multiple samples are to be obtained from a transplant recipient,the frequency of sampling may vary. After samples have begun to be takenfrom a transplant recipient, samples may be obtained about once everyweek, about once every 2 weeks, about once every 3 weeks, about onceevery month, about once every two months, about once every three months,about once every four months, about once every five months, about onceevery six months, about once every year, or about once every two yearsor more after the initial sampling event.

In some embodiments, a transplant recipient has samples of bodily fluidtaken for one to three consecutive months, starting at the one yearanniversary of the transplant event (i.e. 12 months after the transplantevent), providing a total of 4-6 samples for analysis taken over a threemonth time period, with samples being collected about every two weeks.In some embodiments, a transplant recipient has samples of bodily fluidtaken once a week for one to three consecutive months, starting at theone year anniversary of the transplant event (i.e. 12 months after thetransplant event), providing a total of twelve samples for analysistaken over a three month time period. The total duration of obtainingsamples from a transplant recipient, as well as the frequency ofobtaining such samples, may vary and will depend on a variety offactors, such as clinical progress. For example, a transplant recipientmay have samples obtained for analysis of cell-free DNA for the durationof their lifetime. Appropriate timing and frequency of sampling will beable to be determined by one of skill in the art for a given transplantrecipient.

Analysis of Cell-Free DNA in a Transplant Recipient

The methods of the present disclosure involve the analysis of cell-freeDNA from a transplant recipient. After cell-free DNA has been isolatedfrom a transplant recipient, various methods and techniques may be usedto analyze the cell-free DNA. Analysis of cell-free DNA according to themethods of the present disclosure involves analysis of a panel ofpolymorphic markers from the cell-free DNA. In some embodiments, thepolymorphic markers are single nucleotide polymorphisms (SNPs). In someembodiments, SNPs are selected to be included in the panel at least inpart on the basis that the panel of SNPs will be sufficient todifferentiate between donor-derived cell-free DNA and recipient-derivedcell-free DNA.

Panels of Polymorphic Markers

Analysis of cell-free DNA obtained from a transplant recipient involvesthe analysis of a panel of polymorphic markers from the cell-free DNA.Various polymorphic markers may be selected for inclusion in the panelto be analyzed as long as the polymorphic marker panel as a whole issuitable for differentiating between donor-derived cell-free DNA andrecipient-derived cell-free DNA. The same polymorphic marker panel maybe used for each transplant recipient; there is no need to customizepolymorphic marker panels to individualize the panel to differenttransplant recipients.

Various types of polymorphic markers may be included in polymorphicmarker panels. Polymorphic markers are found at a region of DNAcontaining a polymorphism. A polymorphism generally refers to theoccurrence of two or more genetically determined alternative sequencesor alleles in a population. A polymorphic marker or site is the locus atwhich divergence, or the polymorphism, occurs. A polymorphism maycontain, for example, one or more base changes, an insertion, a repeat,or a deletion. A polymorphic locus may be as small as one base pair,such as a SNP. Polymorphic markers may include, for example, singlenucleotide polymorphisms (SNPs), restriction fragment lengthpolymorphisms (RFLPs), short tandem repeats (STRs), variable number oftandem repeats (VNTRs), hypervariable regions, minisatellites,dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats,simple sequence repeats, and insertion elements. Polymorphic markers maycontain one or more bases modified by methylation. Additional types ofpolymorphisms and polymorphic markers will be readily apparent to one ofskill in the art. A polymorphism between two nucleic acids can benaturally occurring, or may be caused by exposure to or contact withchemicals, enzymes, or other agents, or exposure to agents that causedamage to nucleic acids such as, for example, ultraviolet radiation,mutagens, or carcinogens.

Various combinations of polymorphic marker types may be used inpolymorphic marker panels. For example, the polymorphic marker panel mayinclude both SNPs and short tandem repeats, or any other type ofpolymorphic marker. In some embodiments, the polymorphic marker panel iscomposed entirely of SNPs; thus, the polymorphic marker panel is a SNPpanel. Additional combinations of polymorphic markers on polymorphicmarker panels will be readily apparent to one of skill in the art.

Selection of the appropriate quantity and identity of polymorphicmarkers to be analyzed from cell-free DNA may vary, as will beappreciated by one of skill in the art. The panel of polymorphic markersto be analyzed may include at least 10, at least 15, at least 20, atleast 25, at least 30, at least 35, at least 40, at least 45, at least50, at least 55, at least 60, at least 65, at least 70, at least 75, atleast 80, at least 85, at least 90, at least 95, at least 100, at least105, at least 110, or at least 115, at least 120, at least 150, at least200, at least 250, at least 300, at least 350, at least 400, at least450, at least 500, at least 1,000, or at least 1,500 or more independentpolymorphic markers.

In some embodiments, the polymorphic marker panel is a panel of SNPs.SNPs to be included in the SNP panel, or in any other polymorphic markerpanel, may be those previously identified as being suitable fordifferentiating between any two unrelated individuals (Pakstis et al.,2010). For example, the SNP panel may include one or more of thefollowing human SNPs (named according to dbSNP numbering): rs1004357,rs10092491, rs1019029, rs1027895, rs10488710, rs10500617, rs1058083,rs10768550, rs10773760, rs10776839, rs1109037, rs12480506, rs1294331,rs12997453, rs13134862, rs13182883, rs13218440, rs1336071, rs1358856,rs1410059, rs1478829, rs1490413, rs1498553, rs1523537, rs1554472,rs159606, rs1736442, rs1821380, rs1872575, rs2046361, rs2073383,rs214955, rs2175957, rs221956, rs2255301, rs2269355, rs2270529,rs2272998, rs2291395, rs2292972, rs2342747, rs2399332, rs2503107,rs2567608, rs279844, rs2811231, rs2833736, rs2920816, rs315791,rs321198, rs338882, rs3744163, rs3780962, rs4288409, rs430046,rs4364205, rs445251, rs4530059, rs4606077, rs464663, rs4789798,rs4796362, rs4847034, rs521861, rs560681, rs5746846, rs576261, rs590162,rs6444724, rs6591147, rs6811238, rs689512, rs6955448, rs7041158,rs7205345, rs722290, rs7229946, rs740598, rs7520386, rs7704770,rs8070085, rs8078417, rs891700, rs901398, rs9546538, rs9606186,rs985492, rs9866013, rs987640, rs9905977, rs993934, and rs9951171.

SNPs may also be selected on the basis that they have, for example, anoverall population minor allele frequency of >0.4, a target populationminor allele frequency of >0.4, the lowest polymerase error rate (in thetest system) of the 6 potential allele transitions or transversions, andlow linkage on the genome such as, for example, >500 kb distance betweenSNPs.

The SNP panel may include, for example, one or more of the followinghuman SNPs (named according to dbSNP numbering): rs10488710, rs279844,rs1048290, rs1049379, rs1051614, rs1052637, rs1055851, rs1056033,rs1056149, rs1064074, rs1078004, rs10831567, rs6811238, rs11106,rs11210490, rs1126899, rs1127472, rs1127893, rs1130857, rs1049544,rs11547806, rs12237048, rs430046, rs12508837, rs12529, rs12717,rs13184586, rs13295990, rs13428, rs13436, rs1374570, rs14080, rs1411271,rs576261, rs14155, rs1151687, rs1565933, rs1600, rs1678690, rs1881421,rs1897820, rs1898882, rs2056844, rs20575, rs10092491, rs2070426,rs2071888, rs2075322, rs2180314, rs2185798, rs2227910, rs2228560,rs2229571, rs2229627, rs2245285, rs2342747, rs2248490, rs2253592,rs2254357, rs2275047, rs2279665, rs2279776, rs2281098, rs2287813,rs4364205, rs2289751, rs2289818, rs2292830, rs2294092, rs2295005,rs2296545, rs2297236, rs2302443, rs2306049, rs1022478, rs445251,rs230898, rs231235, rs2342767, rs236152, rs2362450, rs2384571,rs2455230, rs246703, rs2480345, rs248385, rs2498982, rs2505232,rs2509943, rs2519123, rs2523072, rs2571028, rs2657167, rs28686812,rs2946994, rs1294331, rs10419826, rs3088241, rs3110623, rs3173615,rs3190321, rs3205187, rs344141, rs35596415, rs362124, rs36657,rs1872575, rs159606, rs3731877, rs3734311, rs3735615, rs3740199,rs3748930, rs3751066, rs3790993, rs3802265, rs3803763, rs1004357,rs3803798, rs3809972, rs3810483, rs3812571, rs3813609, rs3814182,rs3816800, rs3826709, rs3829655, rs3951216, rs1019029, rs408600,rs41317515, rs436278, rs448012, rs475002, rs4845480, rs4849167,rs4865615, rs1027895, rs4890012, rs492594, rs4940019, rs4971514,rs523104, rs528557, rs545500, rs561930, rs57010808, rs57285449,rs10500617, rs6061243, rs609521, rs62490396, rs625223, rs638405,rs6459166, rs648802, rs6510057, rs6764714, rs10768550, rs6790129,rs6794, rs6807362, rs6838248, rs713598, rs7161563, rs726009, rs7289,rs7301328, rs7332388, rs10773760, rs743616, rs743852, rs745142,rs7451713, rs7526132, rs7543016, rs7601771, rs7785899, rs7825,rs8009219, rs10776839, rs8025851, rs8058696, rs8076632, rs8097,rs8103906, rs874881, rs9262, rs9289122, rs936019, rs9393728, rs1109037,rs977070, rs9865242, rs12480506, rs560681, rs12997453, rs13134862,rs13218440, rs1358856, rs1410059, rs1478829, rs1498553, rs1523537,rs4606077, rs1554472, rs1736442, rs1821380, rs2046361, rs214955,rs2175957, rs2255301, rs2269355, rs2270529, rs2272998, rs2291395,rs2292972, rs2399332, rs2503107, rs2567608, rs2811231, rs2833736,rs315791, rs321198, rs6955448, rs338882, rs3780962, rs4288409,rs4530059, rs464663, rs4789798, rs4796362, rs4847034, rs521861,rs1058083, rs5746846, rs590162, rs6444724, rs6591147, rs689512,rs7205345, rs722290, rs740598, rs7520386, rs221956, rs7704770,rs8070085, rs8078417, rs891700, rs901398, rs9546538, rs9606186,rs985492, rs9866013, rs987640, rs13182883, rs9905977, rs993934,rs9951171, rs10274334, rs10421285, rs1043413, rs1044010, rs1045248,rs1045644, and rs1047979. In some embodiments, each of the 266 abovementioned SNPs is included in the polymorphic marker panel to beanalyzed from cell-free DNA.

The SNP panel may include, for example, at least at least 10, at least15, at least 20, at least 25, at least 30, at least 35, at least 40, atleast 45, at least 50, at least 55, at least 60, at least 65, at least70, at least 75, at least 80, at least 85, at least 90, at least 95, atleast 100, at least 105, at least 110, or at least 115, at least 120, atleast 150, at least 200, at least 205, at least 210, at least 215, atleast 220, at least 225, at least 230, at least 235, at least 240, atleast 245, at least 250, at least 255, at least 260, or at least 265 ofthe 266 independent SNPs identified above.

The SNP panel may include, for example, about 10 to about 20, about 20to about 30, about 30 to about 40, about 40 to about 50, about 50 toabout 60, about 60 to about 70, about 70 to about 80, about 80 to about90, about 90 to about 100, about 100 to about 110, about 110 to about120, about 120 to about 130, about 130 to about 140, about 140 to about150, about 150 to about 160, about 160 to about 170, about 170 to about180, about 180 to about 190, about 190 to about 200, about 200 to about210, about 210 to about 220, about 220 to about 230, about 230 to about240, about 240 to about 250, about 250 to about 260, or about 250 toabout 266 of the 266 independent SNPs identified above.

The SNP panel may include, for example, about 195 to about 200, about200 to about 205, about 210 to about 215, about 215 to about 220, about220 to about 225, about 225 to about 230, about 230 to about 235, about235 to about 240, about 240 to about 245, about 245 to about 250, about250 to about 255, about 255 to about 260, about 260 to about 265, orabout 260 to about 266 of the 266 independent SNPs identified above.

Amplification and Sequencing

Cell-free DNA isolated from a transplant recipient may be amplified fordownstream techniques and analysis, such as analysis of a panel ofpolymorphic markers from the cell-free DNA. Various methods andprotocols for DNA extraction are well-known in the art and are describedherein (See e.g. Current Protocols in Molecular Biology, latestedition). Cell-free DNA may be extracted using the QIAamp circulatingnucleic acid kit or other appropriate commercially available kits. Otherexemplary methods of extracting cell-free DNA are well-known (See, e.g.,Cell-Free Plasma DNA as a Predictor of Outcome in Severe Sepsis andSeptic Shock. Clin. Chem. 2008, v. 54, p. 1000-1007; Prediction of MYCNAmplification in Neuroblastoma Using Serum DNA and Real-TimeQuantitative Polymerase Chain Reaction. JCO 2005, v. 23, p. 5205-5210;Circulating Nucleic Acids in Blood of Healthy Male and Female Donors.Clin. Chem. 2005, v. 51, p. 1317-1319; Use of Magnetic Beads for PlasmaCell-free DNA Extraction: Toward Automation of Plasma DNA Analysis forMolecular Diagnostics. Clin. Chem. 2003, v. 49, p. 1953-1955; Chiu R WK, Poon L L M, Lau T K, Leung T N, Wong E M C, Lo Y M D. Effects ofblood-processing protocols on fetal and total DNA quantification inmaternal plasma. Clin Chem 2001; 47: 1607-1613; and Swinkels et al.Effects of Blood-Processing Protocols on Cell-free DNA Quantification inPlasma. Clinical Chemistry, 2003, vol. 49, no. 3, 525-526).

Methods of amplifying DNA are similarly well-known in the art and aredescribed herein. Amplification generally refers to any device, methodor technique that can generate copies of a nucleic acid. Amplificationof cell-free DNA may involve, for example, polymerase chain reaction(PCR) techniques such as linear amplification (cf. U.S. Pat. No.6,132,997), rolling circle amplification, and the like. Cell-free DNAmay be amplified for use in downstream analysis of the DNA by, forexample, digital PCR or sequencing. The Fluidigm Access Array™ System,the RainDance Technologies RainDrop system, or other technologies formultiplex amplification may be used for multiplex or highly parallelsimplex DNA amplification. Amplification may involve the use ofhigh-fidelity polymerases such as, for example, FastStart High Fidelity(Roche), Expand High Fidelity (Roche), Phusion Flash II (ThermoFisherScientific), Phusion Hot Start II (ThermoFisher Scientific), KAPA HiFi(Kapa BioSystems), or KAPA2G (Kapa Biosystems).

Amplification may include an initial PCR cycle that adds a uniquesequence to each individual molecule, called molecular indexing.Molecular indexing allows for quantitative assessment of the absolutelevel of both alleles for each SNP amplicon and therefore may improveprecision and accuracy of determining the percent donor-derivedcell-free DNA.

Amplified DNA may also be subjected to additional processes, such asindexing (also referred to as barcoding or tagging). Methods of indexingDNA are well-known in the art and are described herein. Indexing willallow for the use of multiplex-sequencing platforms, which arecompatible with a variety of sequencing systems, such as Illumina HiSeq,MiSeq, and ThermoFisher Scientific Ion PGM and Ion Proton. Multiplexsequencing permits the sequencing of DNA from multiple samples at oncethrough the use of DNA indexing to specifically identify the samplesource of the sequenced DNA.

The amount of DNA that is used for analysis may vary. In someembodiments, less than 1 pg, 5 pg, 10 pg, 20 pg, 30 pg, 40 pg, 50 pg,100 pg, 200 pg, 500 pg, 1 ng, 5 ng, 10 ng, 20 ng, 30 ng, 40 ng, 50 ng,100 ng, 200 ng, 500 ng, 1 μg, 5 μg, 10 μg, 20 μg, 30 μg, 40 μg, 50 μg,100 μg, 200 μg, 500 μg or 1 mg of DNA are obtained from the sample forfurther genetic analysis. In some cases, about 1-5 pg, 5-10 pg, 10-100pg, 100 pg-1 ng, 1-5 ng, 5-10 ng, 10-100 ng, or 100 ng-1 μg of DNA areobtained from the sample for further genetic analysis.

The methods of the present disclosure involve sequencing target locifrom cell-free DNA, as well as analyzing sequence data. Various methodsand protocols for DNA sequencing and analysis are well-known in the artand are described herein. For example, DNA sequencing may beaccomplished using high-throughput DNA sequencing techniques. Examplesof next generation and high-throughput sequencing include, for example,massively parallel signature sequencing, polony sequencing, 454pyrosequencing, Illumina (Solexa) sequencing with HiSeq, MiSeq, andother platforms, SOLiD sequencing, ion semiconductor sequencing (IonTorrent), DNA nanoball sequencing, heliscope single molecule sequencing,single molecule real time (SMRT) sequencing, MassARRAY®, and DigitalAnalysis of Selected Regions (DANSR™) See, e.g., Stein RA (1 Sep. 2008).“Next-Generation Sequencing Update”. Genetic Engineering & BiotechnologyNews 28 (15); Quail, Michael; Smith, Miriam E; Coupland, Paul; Otto,Thomas D; Harris, Simon R; Connor, Thomas R; Bertoni, Anna; Swerdlow,Harold P; Gu, Yong (1 Jan. 2012). “A tale of three next generationsequencing platforms: comparison of Ion torrent, pacific biosciences andillumina MiSeq sequencers”. BMC Genomics 13 (1): 341; Liu, Lin; Li,Yinhu; Li, Siliang; Hu, Ni; He, Yimin; Pong, Ray; Lin, Danni; Lu, Lihua;Law, Maggie (1 Jan. 2012). “Comparison of Next-Generation SequencingSystems”. Journal of Biomedicine and Biotechnology 2012: 1-11;Qualitative and quantitative genotyping using single base primerextension coupled with matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry (MassARRAY®). Methods Mol Biol. 2009;578:307-43; Chu T, Bunce K, Hogge W A, Peters D G. A novel approachtoward the challenge of accurately quantifying fetal DNA in maternalplasma. Prenat Diagn 2010; 30: 1226-9; and Suzuki N, Kamataki A, YamakiJ, Homma Y. Characterization of circulating DNA in healthy human plasma.Clinica chimica acta; international journal of clinical chemistry 2008;387:55-8). Similarly, software programs for primary and secondaryanalysis of sequence data are well-known in the art.

Where there are multiple cell-free DNA samples from a transplantrecipient to be sequenced, such as when multiple samples are taken fromthe transplant recipient over time, each sample may be sequencedindividually, or multiple samples may be sequenced together usingmultiplex sequencing.

Analyzing Polymorphic Marker Allele Distribution Patterns andDetermining the Level of Donor-Derived Cell-Free DNA

The methods of the present disclosure involve assaying variance inpolymorphic marker allele distribution patterns in a polymorphic markerpanel as compared to expected homozygous or heterozygous distributionpatterns. Analysis of these patterns allows for the determination of thelevel of donor-derived cell-free DNA in a cell-free DNA sample obtainedfrom a transplant recipient.

Generally, an individual contains DNA that is either homozygous orheterozygous for a given polymorphic marker, such as a SNP. Anindividual may be homozygous for one allele of a given polymorphicmarker and will contain 100% of one allele of that polymorphic markerand will contain 0% of the other allele of that polymorphic marker (e.g.100% of allele A for given polymorphic marker, 0% of allele B for givenpolymorphic marker). An individual may also be heterozygous for a givenpolymorphic marker, and thus will contain 50% of allele A and 50% ofallele B of that polymorphic marker. Accordingly, if all of the DNA in asample originated from a single individual, it is expected that anygiven polymorphic marker in the DNA in that sample will exhibit ahomozygous distribution pattern (i.e. 100% of one allele, 0% of theother allele) or a heterozygous distribution pattern (i.e. 50% of oneallele and 50% of the other allele). However, if a DNA sample containsDNA that originated from more than one individual (e.g. a cell-free DNAsample from a transplant recipient that contains both recipient-derivedDNA and donor-derived DNA), then polymorphic marker allele distributionsmay vary, for a given polymorphic marker, from expected homozygous orheterozygous distribution patterns. This is so because two individualsmay not necessarily share the same zygosity for a given polymorphicmarker (e.g. individual 1 is homozygous for a given allele of a givenpolymorphic marker, and individual 2 is heterozygous for the alleles ofthat same polymorphic marker). When this occurs, variance in theexpected allele distribution patterns as compared to expected homozygousor heterozygous distribution patterns may be observed. This variance canbe used to assess whether foreign DNA is present in a DNA sample from asingle individual. With respect to the methods of the presentdisclosure, variance in polymorphic marker allele distribution patternsin the polymorphic marker panel as compared to expected homozygous orheterozygous distribution patterns is used to determine the level ofdonor-derived cell-free DNA in the cell-free DNA sample obtained from atransplant recipient.

When analyzing polymorphic marker sequence data from cell-free DNAaccording to methods of the present disclosure, a majority signal froman allele of a polymorphic marker may be observed and a minority signalfrom an allele of a polymorphic marker may be observed. Regardingcell-free DNA isolated from a transplant recipient, as it is assumedthat the majority of the DNA in the cell-free DNA sample from thetransplant recipient originated from the recipient's own endogenous DNA,it is further assumed that the majority signal represents an allele of apolymorphic marker that originated from recipient-derived cell-free DNA,while the minority signal represents an allele of a polymorphic markerthat originated from donor-derived cell-free DNA. Polymorphic markerssuch as SNPs, for example, with an even distribution of both alleles areassumed to have largely both originated from the recipient. Deviationsfrom the even distribution will indicate the influence of alleles fromthe donor-derived cell-free DNA.

Various calculations may be performed based on allele calls from thesequence data. For example, the methods of the present disclosure mayinvolve calculating various cell-free DNA concentrations, or percentsthereof, of a total amount of cell-free DNA. Overall, as it is assumedthat the majority signal from cell-free DNA in a sample isolated from atransplant recipient is recipient-derived DNA and that the minoritysignal is donor-derived DNA, this information can be used to calculate apercentage of donor-derived DNA in the cell-free DNA sample.

As described above, individual genotyping of the donor and the recipientto determine which allele of the polymorphic marker belongs to the donorand/or the recipient is not necessary, as variance in the polymorphicmarker allele distribution pattern from expected homozygous orheterozygous distribution patterns informs the presence of donor-derivedcell-free DNA in the population of cell-free DNA molecules isolated fromthe transplant recipient. Accordingly, the level of donor-derivedcell-free DNA in a sample obtained from a transplant recipient may bedetermined without using genotype information from the transplantrecipient, from the transplant donor, and/or any other genotypeinformation from any source. Such genotype information that need not beconsidered includes, for example, the genotype across the genome as awhole or portions thereof, or the genotype at the particular polymorphicmarkers being analyzed. In some embodiments, individual genotyping ofthe transplant recipient is not performed. In some embodiments,individual genotyping of the transplant donor is not performed. In someembodiments, neither the transplant recipient nor the transplant donorare individually genotyped. In some embodiments, genotype informationfrom the transplant recipient is not considered when determining thelevels of donor-derived cell-free DNA in a sample obtained from atransplant recipient. In some embodiments, genotype information from thetransplant donor is not considered when determining the levels ofdonor-derived cell-free DNA in a sample obtained from a transplantrecipient. In some embodiments, the levels of donor-derived cell-freeDNA in a sample obtained from a transplant recipient are determinedwithout consideration of genotype information from the transplantrecipient and without consideration of genotype information of thetransplant donor. In some embodiments, the level of donor-derivedcell-free DNA in a sample obtained from a transplant recipient may bedetermined without using genotype information.

Improvements to the calculations may include estimating and subtractinga level of signal due to amplification or sequencing error to improveaccuracy and precision. For example, a suitably chosen subset of SNPsmay be used to estimate a sum, mean, median or standard deviation of thesubset to produce a computation of the overall level of donor-derivedcell-free DNA. Multiple samples from the same subject at a given time ofsampling will all have the same pattern of polymorphic distributionsacross the SNPs, which can be used to enhance the estimate ofdonor-derived cell-free DNA in individual samples from that subject.

The quantity of donor-derived cell-free DNA present in the cell-free DNAsample may be expressed in a variety of ways. In some embodiments, theamount of one or more DNA molecules from donor-derived cell-free DNA isdetermined as a percentage of the total the DNA molecules in the sample.In some embodiments, the amount of one or more DNA molecules fromdonor-derived cell-free DNA is determined as a ratio of the total DNA inthe sample. In some embodiments, the amount of one or more DNA moleculesfrom donor-derived cell-free DNA is determined as a ratio or percentagecompared to one or more reference DNA molecules in the sample. Forexample, the total amount of donor-derived cell-free DNA can bedetermined to be 10% of the total DNA molecules in the cell-free DNAsample. Alternatively, the total amount of donor-derived cell-free DNAcan be at a ratio of 1:10 compared to the total DNA molecules in thecell-free DNA sample. In some embodiments, the amount of one or more DNAmolecules from the donor-derived cell-free DNA can be determined as aconcentration. For example, the total amount of donor-derived cell-freeDNA in the cell-free DNA sample can be determined to be 1 μg/mL. Thevalues described here are merely exemplary to illustrate various ways toexpress quantities of donor-derived cell-free DNA. The percentage ofdonor-derived cell-free DNA in the cell-free DNA sample from atransplant recipient may be extremely low (e.g. at or below 0.5% of thetotal DNA content of the cell-free DNA sample). It is noted that thequantity of recipient-derived cell-free DNA in the cell-free DNA samplemay also be expressed in the manners as described for donor-derivedcell-free DNA. Additional methods of expressing the quantity of a givensource, type, or sequence of DNA molecule in a cell-free DNA sample willbe readily apparent to one of skill in the art.

The above-described embodiments of the present disclosure may beimplemented in a variety of ways. For example, some aspects of theembodiments may be implemented using hardware, software or a combinationthereof. When implemented in software, the software code can be executedon any suitable processor or collection of processors, whether providedin a single computer or distributed among multiple computers. It shouldbe appreciated that any component or collection of components thatperform the functions described above can be generically considered asone or more controllers that control the above-discussed functions. Theone or more controllers can be implemented in numerous ways, such aswith dedicated hardware, or with general-purpose hardware (e.g., one ormore processors) that is programmed using microcode or software toperform the functions recited above.

In this respect, it should be noted that implementation of variousfeatures of the present disclosure may use at least one non-transitorycomputer-readable storage medium (e.g., a computer memory, a floppydisk, a compact disk, a tape, etc.) encoded with a computer program(i.e., a plurality of instructions), which, when executed on aprocessor, performs the above-discussed functions. The computer-readablestorage medium can be transportable such that the program stored thereoncan be loaded onto any computer resource to implement certain aspects ofthe present disclosure discussed herein. In addition, it should be notedthat the reference to a computer program which, when executed, performsthe above-discussed functions, is not limited to an application programrunning on a host computer. Rather, the term computer program is usedherein in a generic sense to reference any type of computer code (e.g.,software or microcode) that can be employed to program a processor toimplement certain aspects of the present disclosure.

Determining Status of an Allograft

The methods of the present disclosure for determining the levels ofdonor-derived cell-free DNA in a sample from a transplant recipient canbe used to determine the status of the allograft in the transplantrecipient. In general, changes in the levels or variance ofdonor-derived cell-free DNA beyond a suitable threshold value in thetransplant recipient over time are informative with regard to the statusof the allograft.

A threshold or threshold value generally refers to any predeterminedlevel or range of levels that is indicative of the presence or absenceof a condition or the presence or absence of a risk. The threshold valuecan take a variety of forms. It can be single cut-off value, such as amedian or mean. As another example, a threshold value can be determinedfrom baseline values before the presence of a condition or risk or aftera course of treatment. Such a baseline can be indicative of a normal orother state in the subject not correlated with the risk or conditionthat is being tested for. For example, the baseline value may be thelevel of donor-derived cell-free DNA in samples from a transplantrecipient prior to the actual transplant event, which would bepresumably zero or negligible, but may also indicate baseline error inthe system. In some embodiments, the threshold value can be a baselinevalue of the subject being tested. The threshold value, as it pertainsto demarcating significant changes in the levels or variance ofdonor-derived cell-free DNA in a transplant recipient, may varyconsiderably. One of skill in the art would recognize appropriateparameters and means for determining significant changes in the levelsor variance of donor-derived cell-free DNA in a transplant recipientover time. Once appropriate analysis parameters are selected,determining changes in the level or variance of donor-derived cell-freeDNA in the transplant recipient over a period of time can inform statusof the allograft.

In some embodiments, an increase in the levels or variance of thedonor-derived cell-free DNA in the transplant recipient over time isindicative of transplant rejection, a need for adjustingimmunosuppressive therapy, immunosuppressive treatment nephrotoxicity,infection, and/or a need for further investigation of the allograftstatus. Without wishing to be bound by theory, it is believed that ifthe level of donor-derived cell-free DNA is increasing in a transplantrecipient over time, then the cells of the allograft are increasinglyexperiencing apoptosis and/or necrosis over time, which is indicative oftransplant rejection.

In some embodiments, a decrease in the levels or variance of thedonor-derived cell-free DNA in the transplant recipient over time isindicative of transplant tolerance, a need for adjustingimmunosuppressive therapy, and/or a need for further investigation ofthe allograft status. Without wishing to be bound by theory, it isbelieved that if the level of donor-derived cell-free DNA is decreasingin a transplant recipient over time, then the cells of the allograft aredecreasingly experiencing apoptosis and/or necrosis over time, which isindicative of transplant tolerance, overimmunosuppression, orappropriate immunosuppression.

In some embodiments, no change in the levels or variance of thedonor-derived cell-free DNA in the transplant recipient over time isindicative of stable transplant rejection status and/or opportunity foradjusting immunosuppressive therapy. Without wishing to be bound bytheory, it is believed that if the level of donor-derived cell-free DNAis not changing in a transplant recipient over time, then the cells ofthe allograft are experiencing a steady-state level of apoptosis overtime, which is indicative of a stable transplant rejection status. Astable transplant rejection status informs the status of the allograftduring the time window analyzed, but does not inform the direction,either towards rejection or tolerance, the allograft is progressingtoward. For example, the allograft may be undergoing active rejection inthe transplant recipient, but a stable transplant rejection statusindicates that the rate of allograft rejection is not changing duringthe time it was analyzed (i.e. the rate of transplant rejection is notincreasing or decreasing). Similarly, the allograft may be undergoingactive tolerance in the transplant recipient, but a stable transplantrejection status indicates that the rate of allograft tolerance is notchanging during the time it was analyzed (i.e. the rate of transplanttolerance is not increasing or decreasing).

Adjustment of Immunosuppressive Therapy

The methods of the present disclosure for determining the levels ofdonor-derived cell-free DNA in a sample from a transplant recipient canbe used to inform the need to adjust immunosuppressive therapy beingadministered to the transplant recipient. In general, changes in thelevels or variance of donor-derived cell-free DNA beyond a suitablethreshold value in the transplant recipient over time are informativewith regard to determining a need to adjust immunosuppressive therapybeing administered to the transplant recipient.

Immunosuppressive therapy generally refers to the administration of animmunosuppressant or other therapeutic agent that suppresses immuneresponses to a subject. Exemplary immunosuppressant agents may include,for example, anticoagulents, antimalarials, heart drugs, non-steroidalanti-inflammatory drugs (NSAIDs), and steroids including, for example,Ace inhibitors, aspirin, azathioprine, B7RP-1-fc, β-blockers, brequinarsodium, campath-1H, celecoxib, chloroquine, corticosteroids, coumadin,cyclophosphamide, cyclosporin A, DHEA, deoxyspergualin, dexamethasone,diclofenac, dolobid, etodolac, everolimus, FK778, feldene, fenoprofen,flurbiprofen, heparin, hydralazine, hydroxychloroquine, CTLA-4 or LFA3immunoglobulin, ibuprofen, indomethacin, ISAtx-247, ketoprofen,ketorolac, leflunomide, meclophenamate, mefenamic acid, mepacrine,6-mercaptopurine, meloxicam, methotrexate, mizoribine, mycophenolatemofetil, naproxen, oxaprozin, Plaquenil, NOX-100, prednisone,methyprenisone, rapamycin (sirolimus), sulindac, tacrolimus (FK506),thymoglobulin, tolmetin, tresperimus, UO126, and antibodies including,for example, alpha lymphocyte antibodies, adalimumab, anti-CD3,anti-CD25, anti-CD52 anti-IL2R, and anti-TAC antibodies, basiliximab,daclizumab, etanercept, hu5C8, infliximab, OKT4, and natalizumab.

In some embodiments, an increase in the levels or variance of thedonor-derived cell-free DNA in the transplant recipient over time isindicative of a need to increase immunosuppressive therapy beingadministered to the transplant recipient. The decision to increaseimmunosuppressive therapy being administered to a transplant recipientmay be based on additional clinical factors, such as the health of thetransplant recipient. In some embodiments, immunosuppressive therapybeing administered to the subject is increased.

In some embodiments, a decrease in the levels or variance of thedonor-derived cell-free DNA in the transplant recipient over time isindicative of a need to decrease immunosuppressive therapy beingadministered to the transplant recipient. The decision to decreaseimmunosuppressive therapy being administered to a transplant recipientmay be based on additional clinical factors, such as the health of thetransplant recipient. In some embodiments, immunosuppressive therapybeing administered to the subject is decreased.

In some embodiments, no change in the levels or variance of thedonor-derived cell-free DNA in the transplant recipient over time isindicative of no need to adjust immunosuppressive therapy beingadministered to the transplant recipient, or that the immunosuppressivetherapy being administered may be maintained. The decision to maintainimmunosuppressive therapy being administered to a transplant recipientmay be based on additional clinical factors, such as the health of thetransplant recipient. In some embodiments, immunosuppressive therapybeing administered to the subject is maintained.

In some embodiments, adjustment of immunosuppressive therapy includeschanging the type or form of immunosuppressant or otherimmunosuppressive therapy being administered to the transplantrecipient. In some embodiments where the transplant recipient is notreceiving immunosuppressive therapy, the methods of the presentdisclosure may indicate a need to begin administering immunosuppressivetherapy to the transplant recipient.

It should be noted that the levels of donor-derived cell-free DNA in thetransplant recipient may not be the only factor taken into considerationwhen determining a need or lack thereof to adjust an immunosuppressivetherapy being administered to the transplant recipient. For example, fora transplant recipient exhibiting both increasing levels ofdonor-derived cell-free DNA over a time interval and increasing severityof an infection, it may not be advisable to increase or even maintainthe current immunosuppressive therapy. It should thus be noted thatimmunosuppressive therapy being administered to a transplant recipientmay be increased, decreased, or maintained irrespective of thedetermined levels of donor-derived cell-free DNA in the transplantrecipient depending on the presence or absence of other controllingclinical factors.

Additional Analyses

The methods of the present disclosure may be performed in addition to orin conjunction with other analyses of samples from a transplantrecipient to diagnose the status of the allograft and/or to inform theneed to adjust immunosuppressive therapy being administered to thesubject.

In some embodiments, the presence or levels of an infectious agent inthe transplant recipient is tested. Infectious agents which may betested for include, for example, viruses; bacteria such as Pseudomonasaeruginosa, Enterobacteriaceae, Nocardia, Streptococcus pneumonia,Staphyloccous aureus, and Legionella; fungi such as Candida,Aspergillus, Cryptococcus, Pneumocystis carinii; or parasites such asToxoplasma gondii.

In some embodiments, the presence or levels of viral infectious agentsin the transplant recipient is tested. Viral biomarkers may be analyzedin nucleic acid obtained from a sample from the transplant recipient todetermine the presence or levels of viruses in the transplant recipient.Viruses which may be tested for include, for example, Cytomegalovirus,Epstein-Barr virus, Anelloviridae, and BK virus. The results of thetests for presence or levels of viruses may be used to classify theimmune status of the transplant recipient and to determine the status ofinfection in the transplant recipient. In some embodiments,immunosuppressive therapies are decreased, or at least not increased, intransplant recipients that are classified as having a high risk ofclinically significant infection. In some embodiments, immunosuppressivetherapies are increased, or at least not decreased, in transplantrecipients that are classified as having a low risk of clinicallysignificant infection. It should be noted that as other clinical factorsmay inform decisions to adjust immunosuppressive therapies, a transplantrecipient may have immunosuppressive therapy currently beingadministered to them increased, decreased, or maintained irrespective ofthe results of tests for presence or levels of viruses and/orclassification for risk of clinically significant infection.

In some embodiments, the methods of the present disclosure also involveperforming an AlloMap test to aid in determining the status of theallograft in a transplant recipient. AlloMap tests involve performingquantitative real-time polymerase chain reaction (qRT-PCR) assays usingRNA that has been isolated from peripheral blood mononuclear cells(PBMC). The expression of a select number of genes is analyzed and thisgene expression data is used to provide information relating to thestatus of an allograft in a transplant recipient. The AlloMap test isknown in the art. Results of an AlloMap test may be used in conjunctionwith the methods of the present disclosure, with or without a method todefine a single score from the combined tests, to determine the statusof an allograft in a transplant recipient and/or inform the need toadjust immunosuppressive therapy being administered to the transplantrecipient.

In some embodiments, the methods of the present disclosure involvedetermining a combination score that may be used to convey the status ofan allograft in a transplant recipient. Combination scores are generallycalculated based on the results of multiple (e.g. two or more) assaysused to probe the status of the allograft in the transplant recipient.For example, combination scores may be calculated based on thedetermined levels of donor-derived cell-free DNA in the transplantrecipient and based on the results of a gene expression profiling assaysuch as, for example, an AlloMap test, which measures select geneexpression. Combination scores may be calculated based on a singlesample taken from a transplant recipient, or they may be based onsamples taken from a transplant recipient over a time interval.Combination scores may be used to determine the status of an allograftin a transplant recipient and/or inform the need to adjustimmunosuppressive therapy being administered to the transplantrecipient.

Additional biomarker analyses, gene expression assays, and other assaysfor diagnosing the status of an allograft in a transplant recipientand/or determining need to adjust immunosuppressive therapy may also beused in addition to or in conjunction with the methods of the presentdisclosure, with or without a method to define a single score from thecombined tests, which will be readily apparent to one of skill in theart.

Additional analysis may be performed to identify markers of new,metastatic, or recurrent cancers in transplant recipients. Primers maybe designed to amplify regions where genetic mutations are known tooccur to provide an early detection of cancer by identification of knowntumor-associated mutations. This may be advantageous at least in partbecause transplant recipients may be at heightened risk of developingcertain malignancies due to overimmunosuppression.

The following Examples are offered to illustrate provided embodimentsand are not intended to limit the scope of the present disclosure.

EXAMPLES Example 1-Analysis of Cell-Free DNA to Determine Status ofTransplanted Organ in a Transplant Recipient and Determine Need toAdjust Immunosuppressive Therapy

This Example demonstrates analysis of samples containing cell-free DNAfrom a transplant recipient to determine the level of donor-derivedcell-free DNA in the samples. Changes in the levels of or variance inthe donor-derived cell-free DNA over time are used to diagnose thestatus of the transplanted organ in the transplant recipient, as well asinform the need to adjust or maintain immunosuppressive therapies beingadministered to the transplant recipient.

Subject Selection

A human patient is selected who was the subject of a kidney transplant12 months prior to this assay as described in this Example. The patientis undergoing treatment with immunosuppressive therapy to preventrejection of the allograft. Separate plasma samples will be collectedfrom this subject on a weekly basis over the course of three consecutivemonths, starting at month 12 (the one year anniversary) after thetransplant event. Accordingly, the transplant recipient will be analyzedduring months 12, 13, and 14 after the transplant event. The methodsdescribed in this Example are applicable to one or more of the samplesisolated from the transplant recipient.

Plasma Collection

Blood is extracted from the subject so that cell-free DNA can beextracted from plasma isolated from this blood sample. The blood sampleis collected in a cell-free DNA blood collection tube (Streck) accordingto the venipuncture method as previously described (Clinical andLaboratory Standards Institute, 2012). The Streck tube is filledcompletely with the blood sample. The tube is removed from the adapterand is immediately mixed by gentle inversion about 8 to 10 times. Aftercollection, the tubes are transported and stored within the temperaturerange of 6-37° C. for up to 14 days. Upon processing of the sample, theStreck tube containing the blood sample is centrifuged at 1600×g for 20minutes at room temperature. The resulting plasma layer is carefullyremoved and is transferred to a 15 mL tube. This plasma sample is thencentrifuged at 1600×g for 10 minutes at room temperature. The resultingplasma layer is carefully removed and placed into a new tube, and theplasma sample may then proceed to have cell-free DNA (cf DNA) extracted.PPT plasma preparation tubes (Becton Dickinson) and CPT pour-off methodsmay also be used during plasma collection.

Cell-Free DNA (cfDNA) Extraction

Approximately 5 mLs of plasma from the plasma sample are used to proceedwith cell-free DNA extraction. For cell-free DNA blood collection tubes(Streck), page 26 of the Qiagen protocol (QIAamp Circulating NucleicAcid Handbook, 2011) may be used with the following modifications: atstep 4 on page 28, the incubation period is 1 hour at 60° C., and step15 on page 29, elute with 30 μL Buffer AVE. For PPT plasma preparationtubes (Becton Dickinson), page 22 of the Qiagen protocol (QIAampCirculating Nucleic Acid Handbook, 2011) may be used with the followingmodifications: at step 15 on page 25, elute with 21 μL Buffer AVE. TheQIAsymphony method may also be used to extract cfDNA. Other DNAextraction methods may also include phenol/chloroform-based extractionmethods.

SNP Selection

After extraction of cell-free DNA from the subject's plasma sample,various SNPs can be assayed in the cell-free DNA. A variety of SNPs areselected for analysis. SNPs may be selected based on those that canprovide the highest possible minor allele frequencies (nearest to 50%).The location of the SNPs in the subject's genome may vary; geneticlinkage may be allowed, but some genetic separation of the SNPs such as,for example, >200 bp separation may also be desirable. The number andidentity of SNPs to be used should provide sufficient power toaccurately estimate the percentage of donor-derived cfDNA present in thesubject's plasma sample. The SNPs to be used in this assay include thosepreviously identified (Pakstis et al., 2010). To amplify the SNPs, 92primer pairs are designed (Fluidigm).

Materials for DNA Amplification

To amplify targeted regions which include SNPs of interest in thecell-free DNA, various materials are assembled which will be used in theamplification process. The FastStart High Fidelity PCR System (Roche)will be used. 92 primer pairs are designed and produced (IDT or Fluidigmper Fluidgim design). ExoI and ExoI buffer (New England BioLabs) will beused. Methods to follow include the Fluidigm pre-amplification protocol(See Page 152 of Access Array System for Illumina Sequencing System).Instruments that will be used include a PCR machine, a plate centrifuge,and a vortexer.

DNA Amplification

The cell-free DNA is amplified according to the Fluidigm Access Arrayprocess and protocols (See page 63 of Access Array System for IlluminaSequencing System). Materials to be used for this amplification protocolinclude a Fluidigm Access Array, or chip, FastStart High Fidelity PCRsystem (Roche), 1×Access Array Harvest Solution (Fluidigm, PN 100-1031),20×Access Array Loading reagent (Fluidigm), and the 92 primer pairsdesigned as described above. Instruments to be used for thisamplification protocol include two IFC Controller AX (Fluidigm) and oneFC1 cycler (Fluidigm).

Barcoding

After cell-free DNA is amplified, the amplified DNA is barcoded.Barcoding may be done, for example, to uniquely identify which of thethree samples any detected amplified DNA originated from if cell-freeDNA molecules from all of the samples are to be sequenced together. Theamplified cell-free DNA is barcoded according to the Fluidigm AccessArray process and protocols (See page 70 of Access Array System forIllumina Sequencing System). Materials to be used for this barcodingprotocol include a FastStart High Fidelity PCR system (Roche), and anAccess Array Barcode Library for Illumina Sequencers (Fluidigm).Instruments that will be used include a PCR machine, a plate centrifuge,and a vortexer.

Sequencing

After the cell-free DNA has been barcoded, it is sequenced. The barcodedcell-free DNA is sequenced according to Fluidigm/Illumina sequencingprotocols for multiplex sequencing (See page 134 of Access Array Systemfor Illumina Sequencing System). Materials to be used for thissequencing protocol include FL1 and FL2 sequencing primers (Fluidigm),HT1 buffer (Illumina), and a MiSeq Reagent Kit v2 (Illumina).Instruments that will be used include a MiSeq sequencing instrument(Illumina).

Data Analysis

After the cell-free DNA is sequenced, it is analyzed to determine thepresence and/or quantity of various SNP alleles as described above.Primary analysis involves the generation of FASTQ output files from theMiSeq instrument. Secondary analysis involves alignment of the outputsequences sequenced by MiSeq to a reference sequence, which in this casewill be sequences from the human genome. The alignment software “Bowtie”is used to conduct the alignment to the whole genome using defaultsettings. If desired, modifications can be made so that only theintended amplicons are aligned. Allele-aware aligners may also be usedto achieve better alignment to non-reference alleles (50% ofalignments). After alignment is complete, variant frequencies areassigned using the “LoFreq” software program (Wilm et al., 2012).Tertiary analysis involves quality control aspects of the analysis. Datais analyzed to ensure that the minimum number of reads have been reachedto achieve sufficient counting. Data is also analyzed to ensure that theminimum number of SNP loci above background and below the heterozygouscall level has been reached. These lower (background) and upper(transplant recipient heterozygous loci) limits may vary. Further,genomic DNA may be determined and a cutoff assigned.

Methods used in the tertiary data will determine the levels ofdonor-derived cell-free DNA in a given sample. The analysis will includedetermining low and high cutoff values, assigning values to homozygousand heterozygous SNPs, multiplying heterozygous SNPs by 2, calculatingthe median, and calculating the confidence interval (CI). Additionalanalysis methods and/or methods to improve analysis quality includelikelihood-based determinations of the contribution of each allele, useof control DNA in each sequencing reaction with defined noise levels perlocus, assigning homozygous or heterozygous status based on likelihoodor β-distributions, and assigning calls that are potentially weighted todeal with compression artifacts at the low end. It is noted that eachadditional sample from a subject may improve the confidence in theaccuracy of determining the percentage of donor-derived cell-free DNA.

Determining Status of the Transplanted Organ

The data analysis methods described above are used to determine thelevel of donor-derived cell-free DNA in each of the cell-free DNAsamples obtained from the transplant recipient. The data analysisinvolves comparison of the levels of donor-derived cell-free DNA in eachof the three samples to determine if the levels of donor-derivedcell-free DNA is increasing, decreasing, or is being maintained atrelatively constant levels in cell-free DNA isolated from the transplantrecipient over time. An increase in the levels or variance of thedonor-derived cell-free DNA over time is indicative of transplantrejection, a need for adjusting immunosuppressive therapy, and/or a needfor further investigation of the transplanted organ status. A decreasein the levels or variance of the donor-derived cell-free DNA over timeis indicative of transplant tolerance, a need for adjustingimmunosuppressive therapy, and/or a need for further investigation ofthe transplanted organ status. No change in the levels or variance ofthe donor-derived cell-free DNA over time is indicative of stabletransplant rejection status and/or an opportunity for adjustingimmunosuppressive therapy.

Example 2—Additional Analysis of Cell-Free DNA to Determine Status ofTransplanted Organ in a Transplant Recipient and Determine Need toAdjust Immunosuppressive Therapy

This Example demonstrates additional analysis of samples containingcell-free DNA from a transplant recipient to determine the level ofdonor-derived cell-free DNA in the samples that builds upon the analysisdescribed in Example 1. Changes in the levels of or variance in thedonor-derived cell-free DNA over time are used to diagnose the status ofthe transplanted organ in the transplant recipient, as well as informthe need to adjust or maintain immunosuppressive therapies beingadministered to the transplant recipient.

Subject Selection

A human patient is selected who was the subject of a kidney transplant12 months prior to this assay as described in this Example. The patientis undergoing treatment with immunosuppressive therapy to preventrejection of the allograft. Separate plasma samples will be collectedfrom this subject on a weekly basis over the course of three consecutivemonths, starting at month 12 (the one year anniversary) after thetransplant event. Accordingly, the transplant recipient will be analyzedduring months 12, 13, and 14 after the transplant event. The methodsdescribed in this Example are applicable to one or more of the samplesisolated from the transplant recipient.

Plasma Collection

Blood is extracted from the subject so that cell-free DNA can beextracted from plasma isolated from this blood sample. The blood sampleis collected in a cell-free DNA blood collection tube (Streck cell-freeDNA BCT) according to the venipuncture method as previously described(Clinical and Laboratory Standards Institute, 2012). The Streck tube isfilled completely with the blood sample. The tube is removed from theadapter and is immediately mixed by gentle inversion about 8 to 10times. After collection, the tubes are transported and stored within thetemperature range of 6-37° C. for up to 14 days. Upon processing of thesample, the Streck tube containing the blood sample is centrifuged at1600×g for 20 minutes at room temperature. The resulting plasma layer iscarefully removed and is transferred to a 15 mL tube. This plasma sampleis then centrifuged at 1600×g for 10 minutes at room temperature, thesupernatant removed and placed into a new tube and centrifuged at16,000×g for 10 minutes at room temperature. The resulting plasma layeris carefully removed and placed into a new tube, and the plasma samplemay then proceed to have cell-free DNA (cf DNA) extracted. PPT plasmapreparation tubes (Becton Dickinson) and CPT pour-off methods may alsobe used during plasma collection.

Cell-Free DNA (cfDNA) Extraction

Approximately 5 mLs of plasma from the plasma sample are used to proceedwith cell-free DNA extraction. For cell-free DNA blood collection tubes(Streck), page 26 of the Qiagen protocol (QIAamp Circulating NucleicAcid Handbook, 2011) may be used with the following modifications: atstep 4 on page 28, the incubation period is 1 hour at 60° C., and step15 on page 29, elute with 30 μL Buffer AVE. For PPT plasma preparationtubes (Becton Dickinson), page 22 of the Qiagen protocol (QIAampCirculating Nucleic Acid Handbook, 2011) may be used with the followingmodifications: at step 15 on page 25, elute with 21 μL Buffer AVE. TheQIAsymphony methods may also be used to extract cfDNA. Other DNAextraction methods may also include phenol/chloroform-based extractionmethods.

SNP Selection

After extraction of cell-free DNA from the subject's plasma sample,various SNPs can be assayed in the cell-free DNA. A variety of SNPs areselected for analysis. SNPs may be selected based on those that canprovide the highest possible minor allele frequencies (nearest to 50%).The location of the SNPs in the subject's genome may vary; geneticlinkage may be allowed, but some genetic separation of the SNPs such as,for example, >200 bp separation may also be desirable. The number andidentity of SNPs to be used should provide sufficient power toaccurately estimate the percentage of donor-derived cfDNA present in thesubject's plasma sample. The SNPs to be used in this assay include thosepreviously identified (Pakstis et al., 2010), as well as others selectedto meet the greater than 0.4 minor allele frequency, an established lowrate of DNA polymerase error, and low linkage. To amplify the SNPs, 266primer pairs are designed (Fluidigm).

DNA Pre-Amplification

To amplify targeted regions which include SNPs of interest in thecell-free DNA, various materials are assembled which will be used in theamplification process. A high-fidelity polymerase such as FastStart HighFidelity (Roche), Expand High Fidelity (Roche), Phusion Flash II(ThermoFisher Scientific), Phusion Hot Start II (ThermoFisherScientific), KAPA HiFi (Kapa BioSystems), or KAPA2G (Kapa Biosystems)will be used. 266 primer pairs are designed and produced (IDT orFluidigm per Fluidgim design). ExoI and ExoI buffer (New EnglandBioLabs) will be used. Methods to follow include the Fluidigmpre-amplification protocol (See Page 152 of Access Array System forIllumina Sequencing System). Instruments that will be used include a PCRmachine, a plate centrifuge, and a vortexer.

DNA Amplification

The cell-free DNA is amplified according to the Fluidigm Access Arrayprocess and protocols (See page 63 of Access Array System for IlluminaSequencing System). Materials to be used for this amplification protocolinclude a Fluidigm Access Array, or chip, a high-fidelity polymerasesuch as FastStart High Fidelity (Roche), Expand High Fidelity (Roche),Phusion Flash II (ThermoFisher Scientific), Phusion Hot Start II(ThermoFisher Scientific), KAPA HiFi (Kapa BioSystems), or KAPA2G (KapaBiosystems), 1× Access Array Harvest Solution (Fluidigm, PN 100-1031),20× Access Array Loading reagent (Fluidigm), and the 226 primer pairsdesigned as described above. Instruments to be used for thisamplification protocol include two IFC Controller AX (Fluidigm) and oneFC1 cycler (Fluidigm).

Indexing (Also Known as Barcoding)

After cell-free DNA is amplified, the amplified DNA is indexed usingindex sequences, also called barcodes or tags. Indexing may be done, forexample, to uniquely identify which of the three samples any detectedamplified DNA originated from if cell-free DNA molecules from all of thesamples are to be sequenced together. The amplified cell-free DNA isindexed according to the Fluidigm Access Array process and protocols(See page 70 of Access Array System for Illumina Sequencing System).Materials to be used for this indexing protocol include a high-fidelitypolymerase such as FastStart High Fidelity (Roche), Expand High Fidelity(Roche), Phusion Flash II (ThermoFisher Scientific), Phusion Hot StartII (ThermoFisher Scientific), KAPA HiFi (Kapa BioSystems), or KAPA2G(Kapa Biosystems), and an Access Array Barcode Library for IlluminaSequencers (Fluidigm—also called an index library). Instruments thatwill be used include a PCR machine, a plate centrifuge, and a vortexer.

Sequencing

After the cell-free DNA has been amplified and indexed, it is sequenced.The indexed cell-free DNA is sequenced according to Fluidigm/Illuminasequencing protocols for multiplex sequencing (See page 134 of AccessArray System for Illumina Sequencing System). Materials to be used forthis sequencing protocol include FL1 and FL2 sequencing primers(Fluidigm), HT1 buffer (Illumina), and a MiSeq Reagent Kit v3(Illumina). Instruments that will be used include a MiSeq sequencinginstrument (Illumina).

Data Analysis

After the cell-free DNA is sequenced, it is analyzed to determine thepresence and/or quantity of various SNP alleles as described above (Seee.g. FIG. 7 ). Primary analysis involves the generation of FASTQ outputfiles from the MiSeq instrument. Secondary analysis involves alignmentof the output sequences sequenced by MiSeq to a reference sequence,which in this case will be sequences from the human genome. End trimminghappens using the “Cutadapt” and “TrimGalore” software packages. Thealignment software “BWA” is used to conduct the alignment to the genomicregions encompassing the set of amplified amplicons. Allele-awarealigners may also be used to achieve better alignment to non-referencealleles (50% of alignments). After alignment is complete, variantfrequencies are assigned using the “SAMtools” software program andsettings customized to minimize inclusion of sequencing errors. Tertiaryanalysis involves quality control aspects of the analysis. Data isanalyzed to ensure that the minimum number of reads have been reached toachieve sufficient counting for each SNP position and ensure that thereare not additional alleles present in the recipient. Data is alsoanalyzed to ensure that the minimum and maximum number of SNP loci abovebackground and below the heterozygous call level has been reached. Theselower (background) and upper (transplant recipient heterozygous loci)limits may vary. In addition, there are metrics to ensure sufficientinput DNA to achieve accurate measurement by determining the quality ofthe heterozygous SNP data. Further, genomic DNA may be determined and acutoff assigned.

Methods used in the tertiary data will determine the levels ofdonor-derived cell-free DNA in a given sample. The analysis includesdetermining the set of SNPs that are homozygous in the recipient,determining low and high cutoff values for the allele frequency of therecipient homozygous SNPs to use in estimating the percent donor-derivedcell-free DNA, computing the mean of the remaining homozygous recipientSNPs, and assessing a multiplier based on the relationship between thedonor and recipient, and calculating the confidence interval (CI).Additional analysis methods and/or methods to improve analysis qualityinclude likelihood- or Bayesian-based estimates of the donor orrecipient allele distributions for each SNP or of the percentdonor-derived cell free DNA, and use of control DNA in each sequencingreaction with defined noise levels per locus. It is noted that eachadditional sample from a subject may improve the confidence in theaccuracy of determining the percentage of donor-derived cell-free DNA.

Determining Status of the Transplanted Organ

The data analysis methods described above are used to determine thelevel of donor-derived cell-free DNA in each of the cell-free DNAsamples obtained from the transplant recipient. The data analysisinvolves comparison of the levels of donor-derived cell-free DNA in eachof the three samples to determine if the levels of donor-derivedcell-free DNA is increasing, decreasing, or is being maintained atrelatively constant levels in cell-free DNA isolated from the transplantrecipient over time. An increase in the levels or variance of thedonor-derived cell-free DNA over time is indicative of transplantrejection, a need for adjusting immunosuppressive therapy, and/or a needfor further investigation of the transplanted organ status. A decreasein the levels or variance of the donor-derived cell-free DNA over timeis indicative of transplant tolerance, a need for adjustingimmunosuppressive therapy, and/or a need for further investigation ofthe transplanted organ status. No change in the levels or variance ofthe donor-derived cell-free DNA over time is indicative of stabletransplant rejection status and/or an opportunity for adjustingimmunosuppressive therapy.

Example 3—Analysis of Cell-Free DNA to Determine Status of TransplantedOrgan in a Heart Transplant Recipient and Predict Need to AdjustImmunosuppressive Therapy

This Example demonstrates analysis of samples containing cell-free DNAfrom a set of 18 transplant recipients to determine the level ofdonor-derived cell-free DNA in the samples. Changes in the levels ofdonor-derived cell-free DNA over time are used to diagnose the status ofthe transplanted organ in the transplant recipient and predict futurestatus of the transplanted organ, as well as inform the need to adjustor maintain immunosuppressive therapies being administered to thetransplant recipient.

Subject Selection

Human patients were selected who were the subject of a heart transplantsprior to this assay as described in this Example. The patients wereundergoing treatment with immunosuppressive therapy to prevent rejectionof the allograft. Separate plasma samples were collected from thesesubjects at visits dictated by the standard of care at their respectivecenters over the course of five consecutive months. The methodsdescribed in this Example are applicable to one or more of the samplesisolated from the transplant recipient.

Plasma Collection

Blood was extracted from the subjects so that cell-free DNA could beextracted from plasma isolated from the blood sample. The blood samplewas collected in PPT tubes according to the venipuncture method aspreviously described (Clinical and Laboratory Standards Institute,2012). The PPT tube (Plasma Preparation Tube, Becton Dickinson) wasfilled completely with the blood sample. The tube was removed from theadapter and immediately mixed by gentle inversion. After collection, thetubes were centrifuged according to the manufacturer's protocol andstored at −80° C. Upon processing of the sample, the tube was thawed andthe plasma layer was carefully removed and transferred to a clean tube.The plasma sample was then centrifuged at 1600×g for 10 minutes at roomtemperature, the supernatant removed and placed into a new tube andcentrifuged at 16,000×g for 10 minutes at room temperature. Theresulting plasma layer was carefully removed and placed into a new tubein preparation to have cell-free DNA (cf DNA) extracted.

Cell-Free DNA (cfDNA) Extraction

Approximately 1 mL of plasma from the plasma sample was used to proceedwith cell-free DNA extraction. For PPT plasma preparation tubes (BectonDickinson), page 22 of the Qiagen protocol (QIAamp Circulating NucleicAcid Handbook, 2011) was used with the following modifications: at step15 on page 25, elute with 21 μL Buffer AVE.

SNP Selection

Various SNPs were selected for analysis to estimate the percentage ofdonor-derived cfDNA present in the subject's plasma sample. The SNPsselected for analysis were rs10488710, rs279844, rs1048290, rs1049379,rs1051614, rs1052637, rs1055851, rs1056033, rs1056149, rs1064074,rs1078004, rs10831567, rs6811238, rs11106, rs11210490, rs1126899,rs1127472, rs1127893, rs1130857, rs1049544, rs11547806, rs12237048,rs430046, rs12508837, rs12529, rs12717, rs13184586, rs13295990, rs13428,rs13436, rs1374570, rs14080, rs1411271, rs576261, rs14155, rs1151687,rs1565933, rs1600, rs1678690, rs1881421, rs1897820, rs1898882,rs2056844, rs20575, rs10092491, rs2070426, rs2071888, rs2075322,rs2180314, rs2185798, rs2227910, rs2228560, rs2229571, rs2229627,rs2245285, rs2342747, rs2248490, rs2253592, rs2254357, rs2275047,rs2279665, rs2279776, rs2281098, rs2287813, rs4364205, rs2289751,rs2289818, rs2292830, rs2294092, rs2295005, rs2296545, rs2297236,rs2302443, rs2306049, rs1022478, rs445251, rs230898, rs231235,rs2342767, rs236152, rs2362450, rs2384571, rs2455230, rs246703,rs2480345, rs248385, rs2498982, rs2505232, rs2509943, rs2519123,rs2523072, rs2571028, rs2657167, rs28686812, rs2946994, rs1294331,rs10419826, rs3088241, rs3110623, rs3173615, rs3190321, rs3205187,rs344141, rs35596415, rs362124, rs36657, rs1872575, rs159606, rs3731877,rs3734311, rs3735615, rs3740199, rs3748930, rs3751066, rs3790993,rs3802265, rs3803763, rs1004357, rs3803798, rs3809972, rs3810483,rs3812571, rs3813609, rs3814182, rs3816800, rs3826709, rs3829655,rs3951216, rs1019029, rs408600, rs41317515, rs436278, rs448012,rs475002, rs4845480, rs4849167, rs4865615, rs1027895, rs4890012,rs492594, rs4940019, rs4971514, rs523104, rs528557, rs545500, rs561930,rs57010808, rs57285449, rs10500617, rs6061243, rs609521, rs62490396,rs625223, rs638405, rs6459166, rs648802, rs6510057, rs6764714,rs10768550, rs6790129, rs6794, rs6807362, rs6838248, rs713598,rs7161563, rs726009, rs7289, rs7301328, rs7332388, rs10773760, rs743616,rs743852, rs745142, rs7451713, rs7526132, rs7543016, rs7601771,rs7785899, rs7825, rs8009219, rs10776839, rs8025851, rs8058696,rs8076632, rs8097, rs8103906, rs874881, rs9262, rs9289122, rs936019,rs9393728, rs1109037, rs977070, rs9865242, rs12480506, rs560681,rs12997453, rs13134862, rs13218440, rs1358856, rs1410059, rs1478829,rs1498553, rs1523537, rs4606077, rs1554472, rs1736442, rs1821380,rs2046361, rs214955, rs2175957, rs2255301, rs2269355, rs2270529,rs2272998, rs2291395, rs2292972, rs2399332, rs2503107, rs2567608,rs2811231, rs2833736, rs315791, rs321198, rs6955448, rs338882,rs3780962, rs4288409, rs4530059, rs464663, rs4789798, rs4796362,rs4847034, rs521861, rs1058083, rs5746846, rs590162, rs6444724,rs6591147, rs689512, rs7205345, rs722290, rs740598, rs7520386, rs221956,rs7704770, rs8070085, rs8078417, rs891700, rs901398, rs9546538,rs9606186, rs985492, rs9866013, rs987640, rs13182883, rs9905977,rs993934, rs9951171, rs10274334, rs10421285, rs1043413, rs1044010,rs1045248, rs1045644, and rs1047979. To amplify the SNPs, 266 primerpairs were designed (Fluidigm).

DNA Pre-Amplification

To amplify targeted regions which include SNPs of interest in thecell-free DNA, various materials were assembled and used in theamplification process. The Phusion Hot Start II DNA Polymerase(ThermoFisher Scientific) was used. 266 primer pairs were designed andproduced (IDT or Fluidigm per Fluidgim design). ExoI and ExoI buffer(New England BioLabs) were used. Methods followed included the Fluidigmpre-amplification protocol (See Page 152 of Access Array System forIllumina Sequencing System). Instruments used included a PCR machine, aplate centrifuge, and a vortexer.

DNA Amplification

The cell-free DNA from the pre-amplification was amplified according tothe Fluidigm Access Array process and protocols (See page 63 of AccessArray System for Illumina Sequencing System). Materials used for thisamplification protocol included a Fluidigm Access Array, or chip, thehigh-fidelity DNA polymerase Phusion Flash II (ThermoFisher Scientific),1× Access Array Harvest Solution (Fluidigm, PN 100-1031), 20× AccessArray Loading reagent (Fluidigm), and the 266 primer pairs designed asdescribed above. Instruments used for this amplification protocolincluded two IFC Controller AX (Fluidigm) and one FC1 cycler (Fluidigm).

Indexing (Also Known as Barcoding)

After cell-free DNA was amplified, the amplified DNA was indexed usingindex sequences, also called barcodes or tags. Indexing may be done, forexample, to uniquely identify which of the three samples any detectedamplified DNA originated from if cell-free DNA molecules from all of thesamples are to be sequenced together. The amplified cell-free DNA wasindexed according to the Fluidigm Access Array process and protocols(See page 70 of Access Array System for Illumina Sequencing System).Materials used for this indexing protocol included the high-fidelitypolymerase Phusion Hot Start II (ThermoFisher Scientific), and an AccessArray Barcode Library for Illumina Sequencers (Fluidigm—also called anindex library). Instruments used included a PCR machine, a platecentrifuge, and a vortexer.

Sequencing

After the cell-free DNA was amplified and indexed, it was sequenced. Theindexed cell-free DNA was sequenced according to Fluidigm/Illuminasequencing protocols for multiplex sequencing (See page 134 of AccessArray System for Illumina Sequencing System). Materials used for thissequencing protocol included FL1 and FL2 sequencing primers (Fluidigm),HT1 buffer (Illumina), and a MiSeq Reagent Kit v3 (Illumina). Cell-freeDNA was sequenced using a MiSeq sequencing instrument (Illumina).

Data Analysis

After the cell-free DNA was sequenced, it was analyzed to determine thepresence and/or quantity of various SNP alleles (See FIG. 7 for generaloutline). Primary analysis involved the generation of FASTQ output filesfrom the MiSeq instrument. Secondary analysis involved alignment of theoutput sequences sequenced by MiSeq to the human genome referencesequence. End trimming was performed using the “Cutadapt” and“TrimGalore” software packages. The alignment software “BWA” was used toconduct the alignment to the genomic regions encompassing the set ofamplified amplicons. After alignment was complete, variant frequencieswere assigned using the “SAMtools” software program and settingscustomized to minimize inclusion of sequencing errors.

Tertiary analysis of this type of data generally involves qualitycontrol aspects of the analysis. Data is analyzed to ensure that theminimum number of reads have been reached to achieve sufficient countingfor each SNP position and ensure that there are not additional allelespresent in the recipient. Data is also analyzed to ensure that theminimum and maximum number of SNP loci above background and below theheterozygous call level has been reached. These lower (background) andupper (transplant recipient heterozygous loci) limits may vary. Inaddition, there are metrics to ensure sufficient input DNA to achieveaccurate measurement by determining the quality of the heterozygous SNPdata. Further, genomic DNA may be determined and a cutoff assigned.

Methods used in the tertiary data determined the levels of donor-derivedcell-free DNA in a given sample. The analysis included adjusting theminor allele frequency of the SNPs for sequencing or amplificationerrors by subtracting an empirically determined error rate for eachtransition or transversion, determining the set of SNPs that have aminor allele frequency lower than a cutoff between 0.1 to 0.25 ashomozygous in the recipient, then using the level of the minor allele inthese SNPs for calculation of donor contribution as a percent of thetotal cell-free DNA. SNPs with values less than 0.0008 minor allelefrequency were removed. The median of the lower 55.4% of the remainingSNPs was doubled and averaged with the median of the highest 44.6% ofthe SNPs to estimate the donor contribution.

Determining Status of the Transplanted Organ

The data analysis methods described above were used to determine thelevel of donor-derived cell-free DNA in each of the cell-free DNAsamples obtained from the transplant recipients. The data analysisinvolved comparison of the levels of donor-derived cell-free DNA in eachof the samples to the other samples from that patient to determine ifthe levels of donor-derived cell-free DNA were increasing, decreasing,or were being maintained at relatively constant levels in cell-free DNAisolated from the transplant recipient over time. An increase in thelevels or variance of the donor-derived cell-free DNA over time isindicative of transplant rejection as shown in FIG. 1 and FIG. 2 . FIG.1 shows the relationship between well-characterized rejection and highpercent donor-derived cell-free DNA. FIG. 2 shows the ability ofelevated cell-free DNA to predict impending rejection withinapproximately one month. This suggests that the physician may need toadjust immunosuppressive therapy, and/or further investigate thetransplanted organ status.

Example 4—Analysis of Cell-Free DNA to Determine Status of TransplantedOrgan in a Transplant Recipient, Adjust Immunosuppressive Therapy, andMonitor Treatment

This Example demonstrates the analysis of samples containing cell-freeDNA from a heart transplant recipient and determination of the level ofdonor-derived cell-free DNA in the samples. The levels of thedonor-derived cell-free DNA are used to diagnose the status of thetransplanted organ in the transplant recipient, as well as inform theneed to adjust or maintain immunosuppressive therapies beingadministered to the transplant recipient. Ongoing changes in the levelsof donor-derived cell-free DNA may be subsequently used to monitorsuccess of the changes in immunosuppressive therapy.

Subject Selection

A human patient was selected who was the subject of a heart transplantprior to this assay as described in this Example. The patient wasundergoing treatment with immunosuppressive therapy to prevent rejectionof the allograft. Separate plasma samples were collected at regularvisits according to the standard of care within the three monthsfollowing rejection. Accordingly, the transplant recipient was analyzedduring the weeks following a rejection event. The methods described inthis Example are applicable to one or more of the samples isolated fromthe transplant recipient.

Plasma Collection

Blood was extracted from the subject so that cell-free DNA could beextracted from plasma isolated from the blood sample. The blood samplewas collected in PPT tubes according to the venipuncture method aspreviously described (Clinical and Laboratory Standards Institute,2012). The PPT tube (Plasma Preparation Tube, Becton Dickinson) wasfilled completely with the blood sample. The tube was removed from theadapter and was immediately mixed by gentle inversion. After collection,the tubes were centrifuged according to the manufacturer's protocol andstored at −80° C. Upon processing of the sample, the tube was thawed andthe plasma layer was carefully removed and transferred to a clean tube.This plasma sample was then centrifuged at 1600×g for 10 minutes at roomtemperature, the supernatant removed and placed into a new tube andcentrifuged at 16,000×g for 10 minutes at room temperature. Theresulting plasma layer was carefully removed and placed into a new tube,and the plasma sample proceeded to have cell-free DNA (cf DNA)extracted.

Cell-Free DNA (cfDNA) Extraction

Approximately 1 mL of plasma from the plasma sample was used to proceedwith cell-free DNA extraction. For PPT plasma preparation tubes (BectonDickinson), page 22 of the Qiagen protocol (QIAamp Circulating NucleicAcid Handbook, 2011) was used with the following modifications: at step15 on page 25, elute with 21 μL Buffer AVE.

SNP Selection

Various SNPs were selected for analysis to estimate the percentage ofdonor-derived cfDNA present in the subject's plasma sample. The SNPsselected for analysis were rs10488710, rs279844, rs1048290, rs1049379,rs1051614, rs1052637, rs1055851, rs1056033, rs1056149, rs1064074,rs1078004, rs10831567, rs6811238, rs11106, rs11210490, rs1126899,rs1127472, rs1127893, rs1130857, rs1049544, rs11547806, rs12237048,rs430046, rs12508837, rs12529, rs12717, rs13184586, rs13295990, rs13428,rs13436, rs1374570, rs14080, rs1411271, rs576261, rs14155, rs1151687,rs1565933, rs1600, rs1678690, rs1881421, rs1897820, rs1898882,rs2056844, rs20575, rs10092491, rs2070426, rs2071888, rs2075322,rs2180314, rs2185798, rs2227910, rs2228560, rs2229571, rs2229627,rs2245285, rs2342747, rs2248490, rs2253592, rs2254357, rs2275047,rs2279665, rs2279776, rs2281098, rs2287813, rs4364205, rs2289751,rs2289818, rs2292830, rs2294092, rs2295005, rs2296545, rs2297236,rs2302443, rs2306049, rs1022478, rs445251, rs230898, rs231235,rs2342767, rs236152, rs2362450, rs2384571, rs2455230, rs246703,rs2480345, rs248385, rs2498982, rs2505232, rs2509943, rs2519123,rs2523072, rs2571028, rs2657167, rs28686812, rs2946994, rs1294331,rs10419826, rs3088241, rs3110623, rs3173615, rs3190321, rs3205187,rs344141, rs35596415, rs362124, rs36657, rs1872575, rs159606, rs3731877,rs3734311, rs3735615, rs3740199, rs3748930, rs3751066, rs3790993,rs3802265, rs3803763, rs1004357, rs3803798, rs3809972, rs3810483,rs3812571, rs3813609, rs3814182, rs3816800, rs3826709, rs3829655,rs3951216, rs1019029, rs408600, rs41317515, rs436278, rs448012,rs475002, rs4845480, rs4849167, rs4865615, rs1027895, rs4890012,rs492594, rs4940019, rs4971514, rs523104, rs528557, rs545500, rs561930,rs57010808, rs57285449, rs10500617, rs6061243, rs609521, rs62490396,rs625223, rs638405, rs6459166, rs648802, rs6510057, rs6764714,rs10768550, rs6790129, rs6794, rs6807362, rs6838248, rs713598,rs7161563, rs726009, rs7289, rs7301328, rs7332388, rs10773760, rs743616,rs743852, rs745142, rs7451713, rs7526132, rs7543016, rs7601771,rs7785899, rs7825, rs8009219, rs10776839, rs8025851, rs8058696,rs8076632, rs8097, rs8103906, rs874881, rs9262, rs9289122, rs936019,rs9393728, rs1109037, rs977070, rs9865242, rs12480506, rs560681,rs12997453, rs13134862, rs13218440, rs1358856, rs1410059, rs1478829,rs1498553, rs1523537, rs4606077, rs1554472, rs1736442, rs1821380,rs2046361, rs214955, rs2175957, rs2255301, rs2269355, rs2270529,rs2272998, rs2291395, rs2292972, rs2399332, rs2503107, rs2567608,rs2811231, rs2833736, rs315791, rs321198, rs6955448, rs338882,rs3780962, rs4288409, rs4530059, rs464663, rs4789798, rs4796362,rs4847034, rs521861, rs1058083, rs5746846, rs590162, rs6444724,rs6591147, rs689512, rs7205345, rs722290, rs740598, rs7520386, rs221956,rs7704770, rs8070085, rs8078417, rs891700, rs901398, rs9546538,rs9606186, rs985492, rs9866013, rs987640, rs13182883, rs9905977,rs993934, rs9951171, rs10274334, rs10421285, rs1043413, rs1044010,rs1045248, rs1045644, and rs1047979. To amplify the SNPs, 266 primerpairs were designed (Fluidigm).

DNA Pre-Amplification

To amplify targeted regions which include SNPs of interest in thecell-free DNA, various materials were assembled and used in theamplification process. The Phusion Hot Start II DNA Polymerase(ThermoFisher Scientific) was used. 266 primer pairs were designed andproduced (IDT or Fluidigm per Fluidigm design). ExoI and ExoI buffer(New England BioLabs) were used. Methods followed included the Fluidigmpre-amplification protocol (See Page 152 of Access Array System forIllumina Sequencing System). Instruments used included a PCR machine, aplate centrifuge, and a vortexer.

DNA Amplification

The cell-free DNA from the pre-amplification was amplified according tothe Fluidigm Access Array process and protocols (See page 63 of AccessArray System for Illumina Sequencing System). Materials used for thisamplification protocol included a Fluidigm Access Array, or chip, thehigh-fidelity DNA polymerase Phusion Flash II (ThermoFisher Scientific),1× Access Array Harvest Solution (Fluidigm, PN 100-1031), 20× AccessArray Loading reagent (Fluidigm), and the 266 primer pairs designed asdescribed above. Instruments used for this amplification protocolincluded two IFC Controller AX (Fluidigm) and one FC1 cycler (Fluidigm).

Indexing (Also Known as Barcoding)

After cell-free DNA was amplified, the amplified DNA was indexed usingindex sequences, also called barcodes or tags. Indexing may be done, forexample, to uniquely identify which of the three samples any detectedamplified DNA originated from if cell-free DNA molecules from all of thesamples are to be sequenced together. The amplified cell-free DNA wasindexed according to the Fluidigm Access Array process and protocols(See page 70 of Access Array System for Illumina Sequencing System).Materials used for this indexing protocol included the high-fidelitypolymerase Phusion Hot Start II (ThermoFisher Scientific), and an AccessArray Barcode Library for Illumina Sequencers (Fluidigm—also called anindex library). Instruments used included a PCR machine, a platecentrifuge, and a vortexer.

Sequencing

After the cell-free DNA was amplified and indexed, it was sequenced. Theindexed cell-free DNA was sequenced according to Fluidigm/Illuminasequencing protocols for multiplex sequencing (See page 134 of AccessArray System for Illumina Sequencing System). Materials used for thissequencing protocol included FL1 and FL2 sequencing primers (Fluidigm),HT1 buffer (Illumina), and a MiSeq Reagent Kit v3 (Illumina). Cell-freeDNA was sequenced using a MiSeq sequencing instrument (Illumina).

Data Analysis

After the cell-free DNA was sequenced, it was analyzed to determine thepresence and/or quantity of various SNP alleles (See FIG. 7 for generaloutline). Primary analysis involved the generation of FASTQ output filesfrom the MiSeq instrument. Secondary analysis involved alignment of theoutput sequences sequenced by MiSeq to the human genome referencesequence. End trimming was performed using the “Cutadapt” and“TrimGalore” software packages. The alignment software “BWA” was used toconduct the alignment to the genomic regions encompassing the set ofamplified amplicons. After alignment was complete, variant frequencieswere assigned using the “SAMtools” software program and settingscustomized to minimize inclusion of sequencing errors.

Tertiary analysis of this type of data generally involves qualitycontrol aspects of the analysis. Data is analyzed to ensure that theminimum number of reads have been reached to achieve sufficient countingfor each SNP position and ensure that there are not additional allelespresent in the recipient. Data is also analyzed to ensure that theminimum and maximum number of SNP loci above background and below theheterozygous call level has been reached. These lower (background) andupper (transplant recipient heterozygous loci) limits may vary. Inaddition, there are metrics to ensure sufficient input DNA to achieveaccurate measurement by determining the quality of the heterozygous SNPdata. Further, genomic DNA may be determined and a cutoff assigned.

Methods used in the tertiary data determined the levels of donor-derivedcell-free DNA in a given sample. The analysis included adjusting theminor allele frequency of the SNPs for sequencing or amplificationerrors by subtracting an empirically determined error rate for eachtransition or transversion, determining the set of SNPs that have aminor allele frequency lower than a cutoff between 0.1 to 0.25 ashomozygous in the recipient, then using the level of the minor allele inthese SNPs for calculation of donor contribution as a percent of thetotal cell-free DNA. SNPs with values less than 0.0008 minor allelefrequency were removed. The median of the lower 55.4% of the remainingSNPs was doubled and averaged with the median of the highest 44.6% ofthe SNPs to estimate the donor contribution.

Determining Status of the Transplanted Organ

The data analysis methods described above were used to determine thelevel of donor-derived cell-free DNA in each of the cell-free DNAsamples obtained from the transplant recipient. The data analysisinvolved comparison of the levels of donor-derived cell-free DNA in eachof the samples to the other samples from that patient to determine ifthe levels of donor-derived cell-free DNA were increasing, decreasing,or were being maintained at relatively constant levels in cell-free DNAisolated from the transplant recipient over time. FIG. 1 shows therelationship between well-characterized rejection and high percentdonor-derived cell-free DNA. In this example, as shown in FIG. 3 , thepatient had experienced rejection (as determined by endomyocardialbiopsy) and also had high levels of dd-cfDNA (>5%). The patient wasgiven a bolus steroid immunosuppressive treatment. Subsequent samplestaken at 20 and 51 days following treatment demonstrate that successfulimmunosuppressive treatment can be monitored by examination of dd-cfDNAlevels for return to levels that indicate no rejection (FIG. 3 ).

Example 5—Analysis of Cell-Free DNA to Determine Status of KidneyTransplant in a Transplant Recipient, Adjust Immunosuppressive Therapy,and Monitor Treatment

This Example demonstrates the analysis of samples containing cell-freeDNA from a kidney transplant recipient to determine the level ofdonor-derived cell-free DNA in the samples. Changes in the levels of orvariance in the donor-derived cell-free DNA over time were used todiagnose the status of the transplanted organ in the transplantrecipient, as well as inform the need to adjust or maintainimmunosuppressive therapies being administered to the transplantrecipient.

Subject Selection

A human patient was selected who was the subject of a kidney transplant8 days prior to this assay as described in this Example. The patient wasundergoing treatment with immunosuppressive therapy to prevent rejectionof the allograft. Separate plasma samples were collected from thissubject for three consecutive months, starting at the first rejectionevent 8 days post-transplant. Accordingly, the transplant recipient wasanalyzed during the weeks following a rejection event. The methodsdescribed in this Example are applicable to one or more of the samplesisolated from the transplant recipient.

Plasma Collection

Blood was extracted from the subject so that cell-free DNA could beextracted from plasma isolated from the blood sample. The blood samplewas collected in CPT tubes according to the venipuncture method aspreviously described (Clinical and Laboratory Standards Institute,2012). The CPT tube (Becton Dickinson) was filled completely with theblood sample. The tube was removed from the adapter and was immediatelymixed by gentle inversion. After collection, the tubes were centrifugedaccording to the manufacturer's protocol and the plasma and mononuclearcells fraction was poured off into a tube containing 5 ml PBS. Thissecond tube was centrifuged to pellet the cells and the plasmasupernatant was retained and stored at −80° C. Upon processing of thesample, the tube was thawed and the plasma sample was then centrifugedat 1600×g for 10 minutes at room temperature, the supernatant removedand placed into a new tube and centrifuged at 16,000×g for 10 minutes atroom temperature. The resulting plasma layer was carefully removed andplaced into a new tube, and the plasma sample then proceeded to havecell-free DNA (cfDNA) extracted.

Cell-Free DNA (cfDNA) Extraction

Approximately 1-2 mLs of plasma from the plasma sample was used toproceed with cell-free DNA extraction. For cell-free DNA bloodcollection tubes (Streck), page 26 of the Qiagen protocol (QIAampCirculating Nucleic Acid Handbook, 2011) was used with the followingmodifications: at step 4 on page 28, the incubation period is 1 hour at60° C., and step 15 on page 29, elute with 30 μL Buffer AVE. For PPTplasma preparation tubes (Becton Dickinson), page 22 of the Qiagenprotocol (QIAamp Circulating Nucleic Acid Handbook, 2011) was used withthe following modifications: at step 15 on page 25, elute with 21 μLBuffer AVE.

SNP Selection

Various SNPs were selected for analysis to estimate the percentage ofdonor-derived cfDNA present in the subject's plasma sample. The SNPsselected for analysis were rs10488710, rs279844, rs1048290, rs1049379,rs1051614, rs1052637, rs1055851, rs1056033, rs1056149, rs1064074,rs1078004, rs10831567, rs6811238, rs11106, rs11210490, rs1126899,rs1127472, rs1127893, rs1130857, rs1049544, rs11547806, rs12237048,rs430046, rs12508837, rs12529, rs12717, rs13184586, rs13295990, rs13428,rs13436, rs1374570, rs14080, rs1411271, rs576261, rs14155, rs1151687,rs1565933, rs1600, rs1678690, rs1881421, rs1897820, rs1898882,rs2056844, rs20575, rs10092491, rs2070426, rs2071888, rs2075322,rs2180314, rs2185798, rs2227910, rs2228560, rs2229571, rs2229627,rs2245285, rs2342747, rs2248490, rs2253592, rs2254357, rs2275047,rs2279665, rs2279776, rs2281098, rs2287813, rs4364205, rs2289751,rs2289818, rs2292830, rs2294092, rs2295005, rs2296545, rs2297236,rs2302443, rs2306049, rs1022478, rs445251, rs230898, rs231235,rs2342767, rs236152, rs2362450, rs2384571, rs2455230, rs246703,rs2480345, rs248385, rs2498982, rs2505232, rs2509943, rs2519123,rs2523072, rs2571028, rs2657167, rs28686812, rs2946994, rs1294331,rs10419826, rs3088241, rs3110623, rs3173615, rs3190321, rs3205187,rs344141, rs35596415, rs362124, rs36657, rs1872575, rs159606, rs3731877,rs3734311, rs3735615, rs3740199, rs3748930, rs3751066, rs3790993,rs3802265, rs3803763, rs1004357, rs3803798, rs3809972, rs3810483,rs3812571, rs3813609, rs3814182, rs3816800, rs3826709, rs3829655,rs3951216, rs1019029, rs408600, rs41317515, rs436278, rs448012,rs475002, rs4845480, rs4849167, rs4865615, rs1027895, rs4890012,rs492594, rs4940019, rs4971514, rs523104, rs528557, rs545500, rs561930,rs57010808, rs57285449, rs10500617, rs6061243, rs609521, rs62490396,rs625223, rs638405, rs6459166, rs648802, rs6510057, rs6764714,rs10768550, rs6790129, rs6794, rs6807362, rs6838248, rs713598,rs7161563, rs726009, rs7289, rs7301328, rs7332388, rs10773760, rs743616,rs743852, rs745142, rs7451713, rs7526132, rs7543016, rs7601771,rs7785899, rs7825, rs8009219, rs10776839, rs8025851, rs8058696,rs8076632, rs8097, rs8103906, rs874881, rs9262, rs9289122, rs936019,rs9393728, rs1109037, rs977070, rs9865242, rs12480506, rs560681,rs12997453, rs13134862, rs13218440, rs1358856, rs1410059, rs1478829,rs1498553, rs1523537, rs4606077, rs1554472, rs1736442, rs1821380,rs2046361, rs214955, rs2175957, rs2255301, rs2269355, rs2270529,rs2272998, rs2291395, rs2292972, rs2399332, rs2503107, rs2567608,rs2811231, rs2833736, rs315791, rs321198, rs6955448, rs338882,rs3780962, rs4288409, rs4530059, rs464663, rs4789798, rs4796362,rs4847034, rs521861, rs1058083, rs5746846, rs590162, rs6444724,rs6591147, rs689512, rs7205345, rs722290, rs740598, rs7520386, rs221956,rs7704770, rs8070085, rs8078417, rs891700, rs901398, rs9546538,rs9606186, rs985492, rs9866013, rs987640, rs13182883, rs9905977,rs993934, rs9951171, rs10274334, rs10421285, rs1043413, rs1044010,rs1045248, rs1045644, and rs1047979. To amplify the SNPs, 266 primerpairs were designed (Fluidigm).

DNA Pre-Amplification

To amplify targeted regions which include SNPs of interest in thecell-free DNA, various materials were assembled and used in theamplification process. The high-fidelity DNA polymerase FastStart HighFidelity (Roche) was used. 266 primer pairs were designed and produced(IDT or Fluidigm per Fluidigm design). ExoI and ExoI buffer (New EnglandBioLabs) were used. Methods followed included the Fluidigmpre-amplification protocol (See Page 152 of Access Array System forIllumina Sequencing System). Instruments used included a PCR machine, aplate centrifuge, and a vortexer.

DNA Amplification

The cell-free DNA from the pre-amplification was amplified according tothe Fluidigm Access Array process and protocols (See page 63 of AccessArray System for Illumina Sequencing System). Materials used for thisamplification protocol included a Fluidigm Access Array, or chip, thehigh-fidelity DNA polymerase FastStart High Fidelity (Roche), 1× AccessArray Harvest Solution (Fluidigm, PN 100-1031), 20× Access Array Loadingreagent (Fluidigm), and the 266 primer pairs designed as describedabove. Instruments used for this amplification protocol included two IFCController AX (Fluidigm) and one FC1 cycler (Fluidigm).

Indexing (Also Known as Barcoding)

After cell-free DNA was amplified, the amplified DNA was indexed usingindex sequences, also called barcodes or tags. Indexing may be done, forexample, to uniquely identify which of the three samples any detectedamplified DNA originated from if cell-free DNA molecules from all of thesamples are to be sequenced together. The amplified cell-free DNA wasindexed according to the Fluidigm Access Array process and protocols(See page 70 of Access Array System for Illumina Sequencing System).Materials used for this indexing protocol included the high-fidelity DNApolymerase FastStart High Fidelity (Roche), and an Access Array BarcodeLibrary for Illumina Sequencers (Fluidigm—also called an index library).Instruments used included a PCR machine, a plate centrifuge, and avortexer.

Sequencing

After the cell-free DNA was amplified and indexed, it was sequenced. Theindexed cell-free DNA was sequenced according to Fluidigm/Illuminasequencing protocols for multiplex sequencing (See page 134 of AccessArray System for Illumina Sequencing System). Materials used for thissequencing protocol included FL1 and FL2 sequencing primers (Fluidigm),HT1 buffer (Illumina), and a MiSeq Reagent Kit v3 (Illumina). Cell-freeDNA was sequenced using a MiSeq sequencing instrument (Illumina).

Data Analysis

After the cell-free DNA was sequenced, it was analyzed to determine thepresence and/or quantity of various SNP alleles (See FIG. 7 for generaloutline). Primary analysis involved the generation of FASTQ output filesfrom the MiSeq instrument. Secondary analysis involved alignment of theoutput sequences sequenced by MiSeq to the human genome referencesequence. End trimming was performed using the “Cutadapt” and“TrimGalore” software packages. The alignment software “BWA” was used toconduct the alignment to the genomic regions encompassing the set ofamplified amplicons. After alignment was complete, variant frequencieswere assigned using the “SAMtools” software program and settingscustomized to minimize inclusion of sequencing errors.

Tertiary analysis of this type of data generally involves qualitycontrol aspects of the analysis. Data is analyzed to ensure that theminimum number of reads have been reached to achieve sufficient countingfor each SNP position and ensure that there are not additional allelespresent in the recipient. Data is also analyzed to ensure that theminimum and maximum number of SNP loci above background and below theheterozygous call level has been reached. These lower (background) andupper (transplant recipient heterozygous loci) limits may vary. Inaddition, there are metrics to ensure sufficient input DNA to achieveaccurate measurement by determining the quality of the heterozygous SNPdata. Further, genomic DNA may be determined and a cutoff assigned.

Methods used in the tertiary data determined the levels of donor-derivedcell-free DNA in a given sample. The analysis included adjusting theminor allele frequency of the SNPs for sequencing or amplificationerrors by subtracting an empirically determined error rate for eachtransition or transversion, determining the set of SNPs that have aminor allele frequency lower than a cutoff between 0.1 to 0.25 ashomozygous in the recipient, then using the level of the minor allelefor calculation of donor contribution as a percent of the majorityallele. The 5% highest and 5% lowest values were removed and the mean ofthe remaining SNP minor allele values calculated. This value estimatesthe heterozygous level for the donor contribution, therefore ismultiplied by two to determine the final estimate of donor contribution.

Determining Status of the Transplanted Organ

The data analysis methods described above were used to determine thelevel of donor-derived cell-free DNA in each of the cell-free DNAsamples obtained from the transplant recipient. The data analysisinvolved comparison of the levels of donor-derived cell-free DNA in eachof the samples to the other samples from that patient to determine ifthe levels of donor-derived cell-free DNA were increasing, decreasing,or were being maintained at relatively constant levels in cell-free DNAisolated from the transplant recipient over time. FIG. 1 shows therelationship between well-characterized rejection and high percentdonor-derived cell-free DNA. In this example, as shown in FIG. 4 , thekidney transplant recipient experienced rejection (as determined byrenal biopsy) and also had high levels of dd-cfDNA (>8%). The patientwas treated by adjustment of immunosuppressive therapy. Subsequentsamples taken at 40 and 69 days following treatment demonstrate thatsuccessful immunosuppressive treatment can be monitored by examinationof dd-cfDNA levels for return to levels that indicate no rejection (lessthan 1% dd-cfDNA, FIG. 4 ).

Example 6—Serial Analysis of Cell-Free DNA to Monitor Status ofTransplanted Organ in a Transplant Recipient

This Example demonstrates the analysis of samples containing cell-freeDNA from heart transplant recipients to determine the level ofdonor-derived cell-free DNA in the samples. Changes in the levels of orvariance in the donor-derived cell-free DNA over time were used todiagnose the status of the transplanted organ in a transplant recipient,as well as inform the need to adjust or maintain immunosuppressivetherapies being administered to a transplant recipient.

Subject Selection

Human patients were selected who were the subject of a heart transplantprior to this assay as described in this Example. The patients wereundergoing treatment with immunosuppressive therapy to prevent rejectionof the allograft, but specific treatment information was blinded at thetime of testing. Patient selection criteria required that they be stablepatients without signs of rejection or other concerns regarding thestatus of the transplanted organ. Separate plasma samples were collectedfrom these subjects during clinical visits according to standard of careprescribed by the physician. The methods described in this Example areapplicable to one or more of the samples isolated from the transplantrecipient.

Plasma Collection

Blood was extracted from the subjects so that cell-free DNA could beextracted from plasma isolated from the blood sample. The blood samplewas collected in a cell-free DNA blood collection tube (Streck cell-freeDNA BCT) according to the venipuncture method as previously described(Clinical and Laboratory Standards Institute, 2012). The Streck tube wasfilled completely with the blood sample. The tube was removed from theadapter and was immediately mixed by gentle inversion about 8 to 10times. After collection, the tubes were transported and stored withinthe temperature range of 6-37° C. for up to 7 days. Upon processing ofthe sample, the Streck tube containing the blood sample was centrifugedat 1600×g for 20 minutes at room temperature. The resulting plasma layerwas carefully removed and was transferred to a 15 mL tube. This plasmasample was then centrifuged at 1600×g for 10 minutes at roomtemperature, the supernatant removed and placed into a new tube andcentrifuged at 16,000×g for 10 minutes at room temperature. Theresulting plasma layer was carefully removed and placed into a new tube,and the plasma sample then proceeded to have cell-free DNA (cfDNA)extracted.

Cell-Free DNA (cfDNA) Extraction

Approximately 5 mLs of plasma from the plasma sample was used to proceedwith cell-free DNA extraction. For cell-free DNA blood collection tubes(Streck), page 26 of the Qiagen protocol (QIAamp Circulating NucleicAcid Handbook, 2011) was used with the following modifications: at step4 on page 28, the incubation period is 1 hour at 60° C., and step 15 onpage 29, elute with 30 μL Buffer AVE.

SNP Selection

Various SNPs were selected for analysis to estimate the percentage ofdonor-derived cfDNA present in the subject's plasma sample. The SNPsselected for analysis were rs10488710, rs279844, rs1048290, rs1049379,rs1051614, rs1052637, rs1055851, rs1056033, rs1056149, rs1064074,rs1078004, rs10831567, rs6811238, rs11106, rs11210490, rs1126899,rs1127472, rs1127893, rs1130857, rs1049544, rs11547806, rs12237048,rs430046, rs12508837, rs12529, rs12717, rs13184586, rs13295990, rs13428,rs13436, rs1374570, rs14080, rs1411271, rs576261, rs14155, rs1151687,rs1565933, rs1600, rs1678690, rs1881421, rs1897820, rs1898882,rs2056844, rs20575, rs10092491, rs2070426, rs2071888, rs2075322,rs2180314, rs2185798, rs2227910, rs2228560, rs2229571, rs2229627,rs2245285, rs2342747, rs2248490, rs2253592, rs2254357, rs2275047,rs2279665, rs2279776, rs2281098, rs2287813, rs4364205, rs2289751,rs2289818, rs2292830, rs2294092, rs2295005, rs2296545, rs2297236,rs2302443, rs2306049, rs1022478, rs445251, rs230898, rs231235,rs2342767, rs236152, rs2362450, rs2384571, rs2455230, rs246703,rs2480345, rs248385, rs2498982, rs2505232, rs2509943, rs2519123,rs2523072, rs2571028, rs2657167, rs28686812, rs2946994, rs1294331,rs10419826, rs3088241, rs3110623, rs3173615, rs3190321, rs3205187,rs344141, rs35596415, rs362124, rs36657, rs1872575, rs159606, rs3731877,rs3734311, rs3735615, rs3740199, rs3748930, rs3751066, rs3790993,rs3802265, rs3803763, rs1004357, rs3803798, rs3809972, rs3810483,rs3812571, rs3813609, rs3814182, rs3816800, rs3826709, rs3829655,rs3951216, rs1019029, rs408600, rs41317515, rs436278, rs448012,rs475002, rs4845480, rs4849167, rs4865615, rs1027895, rs4890012,rs492594, rs4940019, rs4971514, rs523104, rs528557, rs545500, rs561930,rs57010808, rs57285449, rs10500617, rs6061243, rs609521, rs62490396,rs625223, rs638405, rs6459166, rs648802, rs6510057, rs6764714,rs10768550, rs6790129, rs6794, rs6807362, rs6838248, rs713598,rs7161563, rs726009, rs7289, rs7301328, rs7332388, rs10773760, rs743616,rs743852, rs745142, rs7451713, rs7526132, rs7543016, rs7601771,rs7785899, rs7825, rs8009219, rs10776839, rs8025851, rs8058696,rs8076632, rs8097, rs8103906, rs874881, rs9262, rs9289122, rs936019,rs9393728, rs1109037, rs977070, rs9865242, rs12480506, rs560681,rs12997453, rs13134862, rs13218440, rs1358856, rs1410059, rs1478829,rs1498553, rs1523537, rs4606077, rs1554472, rs1736442, rs1821380,rs2046361, rs214955, rs2175957, rs2255301, rs2269355, rs2270529,rs2272998, rs2291395, rs2292972, rs2399332, rs2503107, rs2567608,rs2811231, rs2833736, rs315791, rs321198, rs6955448, rs338882,rs3780962, rs4288409, rs4530059, rs464663, rs4789798, rs4796362,rs4847034, rs521861, rs1058083, rs5746846, rs590162, rs6444724,rs6591147, rs689512, rs7205345, rs722290, rs740598, rs7520386, rs221956,rs7704770, rs8070085, rs8078417, rs891700, rs901398, rs9546538,rs9606186, rs985492, rs9866013, rs987640, rs13182883, rs9905977,rs993934, rs9951171, rs10274334, rs10421285, rs1043413, rs1044010,rs1045248, rs1045644, and rs1047979. To amplify the SNPs, 266 primerpairs were designed (Fluidigm).

DNA Pre-Amplification

To amplify targeted regions which include SNPs of interest in thecell-free DNA, various materials were assembled and used in theamplification process. The Phusion Hot Start II DNA Polymerase(ThermoFisher Scientific) was used. 266 primer pairs were designed andproduced (IDT or Fluidigm per Fluidigm design). ExoI and ExoI buffer(New England BioLabs) were used. Methods followed included the Fluidigmpre-amplification protocol (See Page 152 of Access Array System forIllumina Sequencing System). Instruments used included a PCR machine, aplate centrifuge, and a vortexer.

DNA Amplification

The cell-free DNA from the pre-amplification was amplified according tothe Fluidigm Access Array process and protocols (See page 63 of AccessArray System for Illumina Sequencing System). Materials used for thisamplification protocol included a Fluidigm Access Array, or chip, thehigh-fidelity DNA polymerase Phusion Flash II (ThermoFisher Scientific),1× Access Array Harvest Solution (Fluidigm, PN 100-1031), 20× AccessArray Loading reagent (Fluidigm), and the 266 primer pairs designed asdescribed above. Instruments used for this amplification protocolincluded two IFC Controller AX (Fluidigm) and one FC1 cycler (Fluidigm).

Indexing (Also Known as Barcoding)

After cell-free DNA was amplified, the amplified DNA was indexed usingindex sequences, also called barcodes or tags. Indexing may be done, forexample, to uniquely identify which of the three samples any detectedamplified DNA originated from if cell-free DNA molecules from all of thesamples are to be sequenced together. The amplified cell-free DNA wasindexed according to the Fluidigm Access Array process and protocols(See page 70 of Access Array System for Illumina Sequencing System).Materials used for this indexing protocol included the high-fidelitypolymerase Phusion Hot Start II (ThermoFisher Scientific), and an AccessArray Barcode Library for Illumina Sequencers (Fluidigm—also called anindex library). Instruments used included a PCR machine, a platecentrifuge, and a vortexer.

Sequencing

After the cell-free DNA was amplified and indexed, it was sequenced. Theindexed cell-free DNA was sequenced according to Fluidigm/Illuminasequencing protocols for multiplex sequencing (See page 134 of AccessArray System for Illumina Sequencing System). Materials used for thissequencing protocol included FL1 and FL2 sequencing primers (Fluidigm),HT1 buffer (Illumina), and a MiSeq Reagent Kit v3 (Illumina). Cell-freeDNA was sequenced using a MiSeq sequencing instrument (Illumina).

Data Analysis

After the cell-free DNA was sequenced, it was analyzed to determine thepresence and/or quantity of various SNP alleles (See FIG. 7 for generaloutline). Primary analysis involved the generation of FASTQ output filesfrom the MiSeq instrument. Secondary analysis involved alignment of theoutput sequences sequenced by MiSeq to the human genome referencesequence. End trimming was performed using the “Cutadapt” and“TrimGalore” software packages. The alignment software “BWA” was used toconduct the alignment to the genomic regions encompassing the set ofamplified amplicons. After alignment was complete, variant frequencieswere assigned using the “SAMtools” software program and settingscustomized to minimize inclusion of sequencing errors.

Tertiary analysis of this type of data generally involves qualitycontrol aspects of the analysis. Data is analyzed to ensure that theminimum number of reads have been reached to achieve sufficient countingfor each SNP position and ensure that there are not additional allelespresent in the recipient. Data is also analyzed to ensure that theminimum and maximum number of SNP loci above background and below theheterozygous call level has been reached. These lower (background) andupper (transplant recipient heterozygous loci) limits may vary. Inaddition, there are metrics to ensure sufficient input DNA to achieveaccurate measurement by determining the quality of the heterozygous SNPdata. Further, genomic DNA may be determined and a cutoff assigned.

Methods used in the tertiary data determined the levels of donor-derivedcell-free DNA in a given sample. The analysis included adjusting theminor allele frequency of the SNPs for sequencing or amplificationerrors by subtracting an empirically determined error rate for eachtransition or transversion, determining the set of SNPs that have aminor allele frequency lower than a cutoff between 0.1 to 0.25 ashomozygous in the recipient, then using the level of the minor allele inthese SNPs for calculation of donor contribution as a percent of thetotal cell-free DNA. SNPs with values less than 0.0008 minor allelefrequency were removed. The median of the lower 55.4% of the remainingSNPs was doubled and averaged with the median of the highest 44.6% ofthe SNPs to estimate the donor contribution.

Determining Status of the Transplanted Organ

The data analysis methods described above were used to determine thelevel of donor-derived cell-free DNA in each of the cell-free DNAsamples obtained from the transplant recipients. The data analysisinvolved comparison of the levels of donor-derived cell-free DNA in eachof the samples to the other samples from that patient to determine ifthe levels of donor-derived cell-free DNA were increasing, decreasing,or were being maintained at relatively constant levels in cell-free DNAisolated from the transplant recipient over time. An increase in thelevels or variance of the donor-derived cell-free DNA over time isindicative of transplant rejection as shown in FIG. 1 . FIG. 5 shows thestable nature of percent donor-derived cell-free DNA. This suggests thatthe physician will not need to adjust immunosuppressive therapy in thesestable patients.

Example 7—Analysis of Cell-Free DNA to Determine Status of TransplantedOrgan in a Heart Transplant Recipient in Combination with a GeneExpression Test

This Example demonstrates the analysis of samples containing cell-freeDNA from a set of 55 transplant recipients to determine the level ofdonor-derived cell-free DNA in the samples. In addition, samplescontaining RNA from peripheral blood mononuclear cells were used todetermine the levels of gene expression as measured using AlloMapMolecular Expression Testing. Levels of donor-derived cell-free DNA andgene expression were used to diagnose the status of the transplantedorgan in the transplant recipient and predict future status of thetransplanted organ, as well as inform the need to adjust or maintainimmunosuppressive therapies being administered to the transplantrecipient.

Subject Selection

Human patients were selected who were the subject of a heart transplantprior to this assay as described in this Example. The patients wereundergoing treatment with immunosuppressive therapy to prevent rejectionof the allograft. Separate plasma samples and peripheral bloodmononuclear cell lysates were collected from these subjects at visitsdictated by the standard of care at their respective centers. Themethods described in this Example are applicable to one or more of thesamples isolated from the transplant recipient.

RNA Collection, Processing, and Testing

Peripheral blood mononuclear cells (PBMC) were collected, RNAstabilized, RNA isolated, cDNA created, and cDNA measured by real-timequantitative PCR as described for AlloMap Molecular Expression Testing,an FDA-cleared gene expression profile used to monitor clinically stableheart transplant recipients.

Plasma Collection

Blood was extracted from the subjects so that cell-free DNA could beextracted from plasma isolated from the blood sample. The blood samplewas collected in PPT tubes according to the venipuncture method aspreviously described (Clinical and Laboratory Standards Institute,2012). The PPT tube (Plasma Preparation Tube, Becton Dickinson) wasfilled completely with the blood sample. The tube was removed from theadapter and was immediately mixed by gentle inversion. After collection,the tubes were centrifuged according to the manufacturer's protocol andstored at −80° C. Upon processing of the sample, the tube was thawed andthe plasma layer was carefully removed and transferred to a clean tube.This plasma sample was then centrifuged at 1600×g for 10 minutes at roomtemperature, the supernatant removed and placed into a new tube andcentrifuged at 16,000×g for 10 minutes at room temperature. Theresulting plasma layer was carefully removed and placed into a new tube,and the plasma sample then proceeded to have cell-free DNA (cf DNA)extracted.

Cell-Free DNA (cfDNA) Extraction

Approximately 1 mL of plasma from the plasma sample was used to proceedwith cell-free DNA extraction. For PPT plasma preparation tubes (BectonDickinson), page 22 of the Qiagen protocol (QIAamp Circulating NucleicAcid Handbook, 2011) was used with the following modifications: at step15 on page 25, elute with 21 μL Buffer AVE.

SNP Selection

Various SNPs were selected for analysis to estimate the percentage ofdonor-derived cfDNA present in the subject's plasma sample. The SNPsselected for analysis were rs10488710, rs279844, rs1048290, rs1049379,rs1051614, rs1052637, rs1055851, rs1056033, rs1056149, rs1064074,rs1078004, rs10831567, rs6811238, rs11106, rs11210490, rs1126899,rs1127472, rs1127893, rs1130857, rs1049544, rs11547806, rs12237048,rs430046, rs12508837, rs12529, rs12717, rs13184586, rs13295990, rs13428,rs13436, rs1374570, rs14080, rs1411271, rs576261, rs14155, rs1151687,rs1565933, rs1600, rs1678690, rs1881421, rs1897820, rs1898882,rs2056844, rs20575, rs10092491, rs2070426, rs2071888, rs2075322,rs2180314, rs2185798, rs2227910, rs2228560, rs2229571, rs2229627,rs2245285, rs2342747, rs2248490, rs2253592, rs2254357, rs2275047,rs2279665, rs2279776, rs2281098, rs2287813, rs4364205, rs2289751,rs2289818, rs2292830, rs2294092, rs2295005, rs2296545, rs2297236,rs2302443, rs2306049, rs1022478, rs445251, rs230898, rs231235,rs2342767, rs236152, rs2362450, rs2384571, rs2455230, rs246703,rs2480345, rs248385, rs2498982, rs2505232, rs2509943, rs2519123,rs2523072, rs2571028, rs2657167, rs28686812, rs2946994, rs1294331,rs10419826, rs3088241, rs3110623, rs3173615, rs3190321, rs3205187,rs344141, rs35596415, rs362124, rs36657, rs1872575, rs159606, rs3731877,rs3734311, rs3735615, rs3740199, rs3748930, rs3751066, rs3790993,rs3802265, rs3803763, rs1004357, rs3803798, rs3809972, rs3810483,rs3812571, rs3813609, rs3814182, rs3816800, rs3826709, rs3829655,rs3951216, rs1019029, rs408600, rs41317515, rs436278, rs448012,rs475002, rs4845480, rs4849167, rs4865615, rs1027895, rs4890012,rs492594, rs4940019, rs4971514, rs523104, rs528557, rs545500, rs561930,rs57010808, rs57285449, rs10500617, rs6061243, rs609521, rs62490396,rs625223, rs638405, rs6459166, rs648802, rs6510057, rs6764714,rs10768550, rs6790129, rs6794, rs6807362, rs6838248, rs713598,rs7161563, rs726009, rs7289, rs7301328, rs7332388, rs10773760, rs743616,rs743852, rs745142, rs7451713, rs7526132, rs7543016, rs7601771,rs7785899, rs7825, rs8009219, rs10776839, rs8025851, rs8058696,rs8076632, rs8097, rs8103906, rs874881, rs9262, rs9289122, rs936019,rs9393728, rs1109037, rs977070, rs9865242, rs12480506, rs560681,rs12997453, rs13134862, rs13218440, rs1358856, rs1410059, rs1478829,rs1498553, rs1523537, rs4606077, rs1554472, rs1736442, rs1821380,rs2046361, rs214955, rs2175957, rs2255301, rs2269355, rs2270529,rs2272998, rs2291395, rs2292972, rs2399332, rs2503107, rs2567608,rs2811231, rs2833736, rs315791, rs321198, rs6955448, rs338882,rs3780962, rs4288409, rs4530059, rs464663, rs4789798, rs4796362,rs4847034, rs521861, rs1058083, rs5746846, rs590162, rs6444724,rs6591147, rs689512, rs7205345, rs722290, rs740598, rs7520386, rs221956,rs7704770, rs8070085, rs8078417, rs891700, rs901398, rs9546538,rs9606186, rs985492, rs9866013, rs987640, rs13182883, rs9905977,rs993934, rs9951171, rs10274334, rs10421285, rs1043413, rs1044010,rs1045248, rs1045644, and rs1047979. To amplify the SNPs, 266 primerpairs were designed (Fluidigm).

DNA Pre-Amplification

To amplify targeted regions which include SNPs of interest in thecell-free DNA, various materials were assembled and used in theamplification process. The Phusion Hot Start II DNA Polymerase(ThermoFisher Scientific) was used. 266 primer pairs were designed andproduced (IDT or Fluidigm per Fluidigm design). ExoI and ExoI buffer(New England BioLabs) were used. Methods followed included the Fluidigmpre-amplification protocol (See Page 152 of Access Array System forIllumina Sequencing System). Instruments used included a PCR machine, aplate centrifuge, and a vortexer.

DNA Amplification

The cell-free DNA from the pre-amplification was amplified according tothe Fluidigm Access Array process and protocols (See page 63 of AccessArray System for Illumina Sequencing System). Materials used for thisamplification protocol included a Fluidigm Access Array, or chip, thehigh-fidelity DNA polymerase Phusion Flash II (ThermoFisher Scientific),1× Access Array Harvest Solution (Fluidigm, PN 100-1031), 20× AccessArray Loading reagent (Fluidigm), and the 266 primer pairs designed asdescribed above. Instruments used for this amplification protocolincluded two IFC Controller AX (Fluidigm) and one FC1 cycler (Fluidigm).

Indexing (Also Known as Barcoding)

After cell-free DNA was amplified, the amplified DNA was indexed usingindex sequences, also called barcodes or tags. Indexing may be done, forexample, to uniquely identify which of the three samples any detectedamplified DNA originated from if cell-free DNA molecules from all of thesamples are to be sequenced together. The amplified cell-free DNA wasindexed according to the Fluidigm Access Array process and protocols(See page 70 of Access Array System for Illumina Sequencing System).Materials used for this indexing protocol included the high-fidelitypolymerase Phusion Hot Start II (ThermoFisher Scientific), and an AccessArray Barcode Library for Illumina Sequencers (Fluidigm—also called anindex library). Instruments used included a PCR machine, a platecentrifuge, and a vortexer.

Sequencing

After the cell-free DNA was amplified and indexed, it was sequenced. Theindexed cell-free DNA was sequenced according to Fluidigm/Illuminasequencing protocols for multiplex sequencing (See page 134 of AccessArray System for Illumina Sequencing System). Materials used for thissequencing protocol included FL1 and FL2 sequencing primers (Fluidigm),HT1 buffer (Illumina), and a MiSeq Reagent Kit v3 (Illumina). Cell-freeDNA was sequenced using a MiSeq sequencing instrument (Illumina).

cfDNA Data Analysis

After the cell-free DNA was sequenced, it was analyzed to determine thepresence and/or quantity of various SNP alleles (See FIG. 7 for generaloutline). Primary analysis involved the generation of FASTQ output filesfrom the MiSeq instrument. Secondary analysis involved alignment of theoutput sequences sequenced by MiSeq to the human genome referencesequence. End trimming was performed using the “Cutadapt” and“TrimGalore” software packages. The alignment software “BWA” was used toconduct the alignment to the genomic regions encompassing the set ofamplified amplicons. After alignment was complete, variant frequencieswere assigned using the “SAMtools” software program and settingscustomized to minimize inclusion of sequencing errors.

Tertiary analysis of this type of data generally involves qualitycontrol aspects of the analysis. Data is analyzed to ensure that theminimum number of reads have been reached to achieve sufficient countingfor each SNP position and ensure that there are not additional allelespresent in the recipient. Data is also analyzed to ensure that theminimum and maximum number of SNP loci above background and below theheterozygous call level has been reached. These lower (background) andupper (transplant recipient heterozygous loci) limits may vary. Inaddition, there are metrics to ensure sufficient input DNA to achieveaccurate measurement by determining the quality of the heterozygous SNPdata. Further, genomic DNA may be determined and a cutoff assigned.

Methods used in the tertiary data determined the levels of donor-derivedcell-free DNA in a given sample. The analysis included adjusting theminor allele frequency of the SNPs for sequencing or amplificationerrors by subtracting an empirically determined error rate for eachtransition or transversion, determining the set of SNPs that have aminor allele frequency lower than a cutoff between 0.1 to 0.25 ashomozygous in the recipient, then using the level of the minor allele inthese SNPs for calculation of donor contribution as a percent of thetotal cell-free DNA. SNPs with values less than 0.0008 minor allelefrequency were removed. The median of the lower 55.4% of the remainingSNPs was doubled and averaged with the median of the highest 44.6% ofthe SNPs to estimate the donor contribution.

Determining Status of the Transplanted Organ

The data analysis methods described above were used to determine thelevel of donor-derived cell-free DNA in each of the cell-free DNAsamples obtained from the transplant recipients. The data analysisinvolved comparison of the levels of donor-derived cell-free DNA in eachof the samples to the other samples from that patients to determine ifthe levels of donor-derived cell-free DNA was increasing, decreasing, orwas being maintained at relatively constant levels in cell-free DNAisolated from the transplant recipients over time. An increase in thelevels or variance of the donor-derived cell-free DNA over time isindicative of transplant rejection as shown in FIG. 1 and FIG. 6A. Thesetwo figures show the relationship between well-characterized rejectionand high percent donor-derived cell-free DNA. FIG. 6B shows therelationship between well-characterized rejection and the results ofAlloMap Molecular Expression Testing for the gene expression signature.FIG. 6C shows the ability of a combined result from donor-derivedcell-free DNA and gene expression to better discriminate betweenrejection and non-rejection. The two values (percent dd-cfDNA andAlloMap) were scaled to the same range and then additively combined tocreate a single score. This suggests that the physician will have betterinformation about the status of the transplanted organ if both cfDNA andgene expression are used and combined in this way or similar methods.

REFERENCES

-   Clinical and Laboratory Standards Institute. H3-A6, (2012)    Procedures for the Collection of Diagnostic Blood Specimens by    Venipuncture; Approved Standard-Sixth Edition, Vol. 27, No. 26.-   QIAamp Circulating Nucleic Acid Handbook, (2011), Second Edition.-   Pakstis A J, Speed W C, Fang R, Hyland F C, Furtado M R, Kidd J R,    Kidd K K. (2010) SNPs for a universal individual identification    panel. Hum Genet; 127(3):315-24.-   Access Array System for Illumina Sequencing System, P/N 100-3770,    Rev. G1.-   Andreas Wilm, Pauline Poh Kim Aw, Denis Bertrand, Grace Hui Ting    Yeo, Swee Hoe Ong, Chang Hua Wong, Chiea Chuen Khor, Rosemary    Petric, Martin Lloyd Hibberd and Niranjan Nagarajan. (2012) LoFreq:    A sequence-quality aware, ultra-sensitive variant caller for    uncovering cell-population heterogeneity from high-throughput    sequencing datasets. Nucleic Acids Res. 40(22):11189-201.

What is claimed is:
 1. A method of quantifying an amount of non-self DNAin a transplant recipient of pancreatic islet cells from a transplantdonor without consideration of genotype information from the transplantrecipient or donor, the method comprising: (a) providing DNA from asample obtained from the recipient post-transplantation; (b) amplifying,in a targeted manner from the DNA, each single nucleotide polymorphism(SNP) in a panel of two or more SNPs, (c) sequencing the amplified SNPsto obtain homozygous or heterozygous allele distribution patterns of theSNPs (SNP allele distribution patterns) in the panel; and (d)quantifying, without consideration of genotype information from thetransplant donor or recipient, the amount of non-self DNA by assayingvariance in homozygous or heterozygous allele distribution patterns ofthe SNPs in the panel as compared to expected homozygous or heterozygousallele distribution patterns, wherein individual genotyping of the donorand the recipient across the genome as a whole or portions thereof todetermine which allele of each SNP in the panel belongs to the donor andthe recipient is not performed.
 2. The method of claim 1, wherein theDNA is cell-free DNA.
 3. The method of claim 1, wherein the DNA iscell-bound DNA.
 4. The method of claim 2, wherein an amount of non-selfcell-free DNA above a predetermined threshold indicates a status ofexhaustion, contraction, loss of persistence, transplanted cellrejection, disease relapse, and/or graft vs. host disease, and an amountof non-self cell-free DNA below a predetermined threshold indicates astatus of engraftment, expansion and/or persistence of the transplantedcells.
 5. The method of claim 3, wherein an amount of non-selfcell-bound DNA above a predetermined threshold and/or increasing orstable over a time interval indicates a status of engraftment,expansion, and/or persistence of the transplanted cells, and an amountof non-self cell-bound DNA below a predetermined threshold and/ordecreasing over a time interval indicates a status of exhaustion,contraction, loss of persistence, transplanted cell rejection, diseaserelapse, and/or graft vs. host disease.
 6. The method of claim 1,wherein each SNP in the panel is selected to have greater than 0.4 minorallele frequency and low linkage with each other.
 7. The method of claim1, further comprising testing for the presence of an infectious agent.8. The method of claim 7, wherein the infectious agent is selected fromthe group consisting of viruses, bacteria, fungi, and parasites.
 9. Themethod of claim 1, further comprising conducting one or more geneexpression profiling assays.
 10. A kit for carrying out the method ofclaim 1, comprising: primers, reagents, controls for targeted singlenucleotide polymorphism (SNP) amplification and sequencing, instructionsfor use, and instructions for accessing and using software forquantifying an amount of non-self DNA in a transplant recipient ofpancreatic islet cells from a transplant donor.
 11. A method ofquantifying an amount of DNA molecules from non-self DNA in a transplantrecipient of pancreatic islet cells from a transplant donor withoutconsideration of genotype information from the transplant recipient ordonor, the method comprising: (a) providing DNA from a sample obtainedfrom the recipient post-transplantation; (b) amplifying, in a targetedmanner from the DNA, each single nucleotide polymorphism (SNP) in apanel of two or more SNPs, (c) sequencing the amplified SNPs to obtainhomozygous or heterozygous allele distribution patterns of the SNPs (SNPallele distribution patterns) in the panel; and (d) quantifying, withoutconsideration of genotype information from the transplant donor orrecipient, the amount of DNA molecules from non-self DNA by assayingvariance in homozygous or heterozygous allele distribution patterns ofthe SNPs in the panel as compared to expected homozygous or heterozygousallele distribution patterns, wherein individual genotyping of the donorand the recipient across the genome as a whole or portions thereof todetermine which allele of each SNP in the panel belongs to the donor andthe recipient is not performed.
 12. The method of claim 11, wherein theDNA is cell-free DNA.
 13. The method of claim 11, wherein the DNA iscell-bound DNA.
 14. The method of claim 12, wherein an amount of DNAmolecules from non-self cell-free DNA above a predetermined thresholdindicates a status of exhaustion, contraction, loss of persistence,transplanted cell rejection, disease relapse, and/or graft vs. hostdisease, and an amount of DNA molecules from non-self cell-free DNAbelow a predetermined threshold indicates a status of engraftment,expansion and/or persistence of the transplanted cells.
 15. The methodof claim 13, wherein an amount of DNA molecules from non-self cell-boundDNA above a predetermined threshold and/or increasing or stable over atime interval indicates a status of engraftment, expansion, and/orpersistence of the transplanted cells, and an amount of DNA moleculesfrom non-self cell-bound DNA below a predetermined threshold and/ordecreasing over a time interval indicates a status of exhaustion,contraction, loss of persistence, transplanted cell rejection, diseaserelapse, and/or graft vs. host disease.
 16. The method of claim 11,wherein each SNP in the panel is selected to have greater than 0.4 minorallele frequency and low linkage with each other.
 17. The method ofclaim 11, further comprising testing for the presence of an infectiousagent.
 18. The method of claim 17, wherein the infectious agent isselected from the group consisting of viruses, bacteria, fungi, andparasites.
 19. The method of claim 11, further comprising conducting oneor more gene expression profiling assays.
 20. The method of claim 11,further comprising adding at least one reference DNA molecule to thesample before the amplifying step and quantifying an amount of DNAmolecules from the non-self DNA in comparison to the at least onereference DNA molecule.
 21. The method of claim 20, wherein the amountof DNA molecules from the non-self DNA is quantified as copies of DNAmolecules per volume.
 22. A kit for carrying out the method of claim 11,comprising: primers, reagents, controls for targeted single nucleotidepolymorphism (SNP) amplification and sequencing, instructions for use,and instructions for accessing and using software for quantifying anamount of DNA molecules from non-self DNA in a transplant recipient ofpancreatic islet cells from a donor.
 23. A method of preparing amplifiedSNPs useful for quantifying an amount of non-self DNA in a transplantrecipient of pancreatic islet cells from a transplant donor withoutconsideration of genotype information from the transplant recipient ordonor, the method comprising: (a) providing DNA from a sample obtainedfrom the recipient post-transplantation; (b) extracting DNA from thesample, wherein the DNA comprises non-self DNA and recipient-derivedDNA; (c) amplifying, in a targeted manner from the DNA, each singlenucleotide polymorphism (SNP) in a panel of two or more SNPs; (d)sequencing the amplified SNPs to obtain homozygous or heterozygousallele distribution patterns of the SNPs (SNP allele distributionpatterns) in the panel; and (e) quantifying, without consideration ofgenotype information from the transplant donor or recipient, the amountof non-self DNA by assaying variance in homozygous or heterozygousallele distribution patterns of the SNPs in the panel as compared toexpected homozygous or heterozygous allele distribution patterns,wherein individual genotyping of the donor and the recipient across thegenome as a whole or portions thereof to determine which allele of eachSNP in the panel belongs to the donor and the recipient is notperformed.
 24. The method of claim 23, wherein the DNA is cell-free DNA.25. The method of claim 23, wherein the DNA is cell-bound DNA.
 26. Themethod of claim 24, wherein an amount of non-self cell-free DNA above apredetermined threshold indicates a status of exhaustion, contraction,loss of persistence, transplanted cell rejection, disease relapse,and/or graft vs. host disease, and an amount of non-self cell-free DNAbelow a predetermined threshold indicates a status of engraftment,expansion and/or persistence of the transplanted cells.
 27. The methodof claim 25, wherein an amount of non-self cell-bound DNA above apredetermined threshold and/or increasing or stable over a time intervalindicates a status of engraftment, expansion, and/or persistence of thetransplanted cells, and an amount of non-self cell-bound DNA below apredetermined threshold and/or decreasing over a time interval indicatesa status of exhaustion, contraction, loss of persistence, transplantedcell rejection, disease relapse, and/or graft vs. host disease.
 28. Themethod of claim 23, further comprising testing for the presence of aninfectious agent.
 29. The method of claim 28, wherein the infectiousagent is selected from the group consisting of viruses, bacteria, fungi,and parasites.
 30. The method of claim 23, further comprising conductingone or more gene expression profiling assays.